Tetanus toxin functional fragment antigen and tetanus vaccine

ABSTRACT

Disclosed is a tetanus toxin functional fragment antigen (FFA), comprising at least one fragment which is substantially the same as that obtained by a process comprising the steps of splitting at least one peptide bond selected from peptide bonds individually connecting mutually adjacent amino acid residues in a partial amino acid sequence between two cysteine residues participating in forming a disulfide bridge present in the N-terminal of the entire amino acid sequence of the whole tetanus toxin molecule, splitting the disulfide bridge, and splitting non-covalent bonds between groups on the tetanus toxin molecule; wherein the tetanus toxin FFA has a molecular weight of from 90,000 to 110,000 as measured by an SDS-polyacrylamide gel electrophoresis method, and an isoelectric point of 7.25±0.5 as measured by an isoelectric focusing method. The FFA of the present invention not only has a satisfactory immunopotency as a tetanus vaccine antigen, that is, has an immunopotency which is substantially the same as that of a whole tetanus toxin toxoid, but is also extremely excellent with respect to the diminution of adverse side effects. Further, the present invention provides a method for mass production of the FFA, and a tetanus vaccine comprising the FFA, and a combined vaccine comprising the tetanus vaccine and at least one vaccine other than the tetanus vaccine.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to a tetanus toxin functional fragment antigen and a tetanus vaccine comprising the same. More particularly, the present invention is concerned with a specific tetanus toxin functional fragment antigen which is extremely useful as an antigen for a tetanus vaccine since the functional fragment antigen is advantageous not only in that it is extremely excellent with respect to the diminution of side effects when used as an antigen, as compared to the current tetanus vaccine comprising as an antigen a whole tetanus toxin toxoid, but also in that it has an immunopotency which is substantially the same as that of the whole tetanus toxin toxoid. The present invention is also concerned with a very safe and effective tetanus vaccine (tetanus toxoid) comprising the tetanus toxin functional fragment antigen as an active component, a combined vaccine comprising the tetanus vaccine and at least one vaccine other than the tetanus vaccine, and methods for producing the fragment antigen and vaccines.

2. Prior Art

As is well known, tetanus is an infectious disease with extremely high mortality which produces serious symptoms, such as opisthotonos and dyspnea. Tetanus bacilli are widely distributed in the environments, and their spores are commonly found in soil, feces of animals, and the like. Therefore, every individual is exposed to the danger of tetanus infection from various types of traumas, such as punctured wounds and crushed wounds. Moreover, when an individual is infected with tetanus bacilli, conventional chemotherapies using antibiotics, muscle relaxants and the like cannot grossly change the mortality, whether tetanus patients are elderly or young. In developed countries, most deaths from tetanus have recently occurred among the elderly patients who escaped from vaccination against tetanus in their babyhood.

Further, even when an individual receives tetanus vaccination in baby- or child-hood for basal immunization and receives a booster, the immunity remaining in adulthood is not sufficient for preventing tetanus infection when the individual suffers unexpected injury in earthquakes, fires, traffic accidents or the like. Therefore, it is important for adults of the ages above ca. 40, especially elderly persons, to receive personally a booster injection in order to ensure protection against tetanus.

In developed countries, an increase in the number of intrahospital childbirths, and improvements in living environments and sanitation, an improvement in the quality of emergency medical care with respect to the provision of toxoids and antitoxins, and compulsory vaccinations for younger people, have reduced the number of tetanus patients to {fraction (1/30)} of that of half a century ago. Furthermore, tetanus is a non-epidemic disease and is not transmitted from person to person. Therefore, the importance of the prevention of this disease tends to be overlooked. However, even today, the number of tetanus deaths in the world is estimated to be about 1 million per year, including mostly neonatal tetanus deaths which are prevailing in developing countries. In addition, due to widespread drug abuse, the number of tetanus patients infected through contaminated injection needles is also increasing recently.

Under these circumstances, tetanus is now recognized as a disease to be prevented by vaccination, rather than to be treated, and preventive measures against tetanus are being actively undertaken. For example, in the Expanded Program of Immunization (EPI) of the World Health Organization (WHO), vaccination against tetanus is being adopted as one of the most important tasks, and the vaccination program is being promoted. The “International Conference on Tetanus”, one of whose goals is to eradicate the tetanus disease has been held about every three years in various countries since 1963.

As evident from the above, tetanus is a disease caused by a ubiquitous bacteria whose spores are impossible to eradicate from the earth, and it is not an exaggeration to say that vaccination against tetanus is the only way to reduce the death of human beings due to tetanus, irrespective of age and sex, to zero, and that the vaccination is essential for all human beings who are born on the earth not only at present, but also in the future.

For prevention of tetanus, tetanus toxoid has been used as a vaccine. Tetanus toxoid, which is used as an active component for tetanus vaccine, is tetanus toxin detoxified with formalin. Such a tetanus toxoid has been used in either a plain form without an adjuvant or in the form of a precipitated antigen preparation adsorbed on a small amount of an aluminum salt as an adjuvant or in the form of a combined antigen preparation prepared by mixing tetanus toxoid with other vaccines, such as diphtheria toxoid, pertussis vaccine and Haemophilus influenzae b vaccine. To infants, tetanus toxoid is generally administered in the form of the so-called DPT combined vaccine which is a mixture of vaccines of diphtheria (D), tetanus (T) and pertussis (P) in adsorbed forms. For a tetanus-prophylactic treatment of traumatic patients, a plain T toxoid vaccine or a DT combined toxoid vaccine is used. These toxoids are widely used over the world and the T toxoid preparations have been highly appreciated in the world as one of the most effective and important vaccines. However, the current tetanus toxoid preparations have various problems to be solved. For example, the tetanus toxoid has disadvantages in that there are various adverse side effects, that the product quality is uneven among different manufacturers, that the retention of immunity is limited to only approximately 5 to 10 years and, therefore, repeated vaccinations are necessary to keep the antitoxin level sufficient to prevent tetanus infection. Thus, the conventional tetanus toxoid has problems to be solved with respect to safety, control of quality, retention of immunity, and ease, labor saving and economy in administration. Therefore, for promoting the use of the tetanus vaccine, a large number of problems need to be solved mainly from a viewpoint of mass-production of high quality tetanus vaccine.

Hereinbelow, prior art is discussed in connection with the primary object of the present invention, which is to provide a tetanus toxin antigen, which is not only extremely excellent with respect to the diminution of adverse side effects when used as a vaccine, but also exhibits high immunopotency, thus solving the above-mentioned problems accompanying the prior art.

Various adverse side effects are known to accompany the use of conventional tetanus toxoid vaccines. Various adverse side effects, such as local reactions at injection sites (e.g., erythema, tenderness, swelling, edema and sterile abscess), systemic fever; and, although rare, allergy (e.g., local anaphylaxis, anaphylactic shock, serum sickness-like type III hypersensitivity and delayed hypersensitivity) and serious generalized reactions (e.g., peripheral neuropathy, lymphadenopathy, brachial plexus neuropathy, Guillain-Barret syndrome and acute transverse myelitis) have been reported (see “Vaccine”, 2nd edition, edited by S. A. Plotkin and E. A. Mortimer, pp. 75-77, W. B. Saunders Company, 1994; “Mandell, Douglas and Bennett's Principles and Practice of Infectious Diseases”, 4th edition, edited by G. L. Mandell et al., p. 2781, Churchill Livingstone & Son, Inc., 1995; Journal of the American Medical Association, 264(18), p. 2448, 1990 and 271(20), p. 1629, 1994; and Lancet, 339, pp. 1111-1112, May 2, 1992).

Various attempts to reduce or remove these adverse side effects of the tetanus toxoid vaccine have been made. For example, development of a method for obtaining highly purified toxoid, use of modified or new adjuvants, and individual use of the fragments A, B and C (which are subunits of the tetanus toxin and which are explained below) as an active component for a vaccine have been proposed. Of these attempts, with respect to the techniques of using a tetanus toxin fragment, as examples of tetanus toxin fragments used in these techniques, there can be mentioned fragment A or C prepared by digesting the tetanus toxin with trypsin and/or papain (see Unexamined Japanese Patent Application Laid-Open Specification Nos. 50-71820, 51-82719 and 52-83928), fragment A-B prepared by digesting the tetanus toxin with papain (see Unexamined Japanese Patent Application Laid-Open Specification No. 53-26319), an antigen obtained by expressing a gene coding for fragment C in E. coli, yeast or salmonella (see Unexamined Japanese Patent Application Laid-Open Specification No. 3-285681, Japanese Patent Application prior-to-examination Publication (Kohyo) No. 4-506005, corresponding to International Application Publication No. WO 90/15871, International Application Publication No. WO 94/03615, and EP-A-0 209 281), and a synthesized epitope of fragment C (see International Application Publication No. WO 94/00484). However, none of these conventional tetanus toxin fragment vaccines have been put into practical use because all of these tetanus toxin fragment vaccines have low antigenicity and immunopotency, as compared to those of the conventional tetanus toxoid comprising the toxoid of the whole tetanus toxin molecule. Meanwhile, cloning of the tetanus toxin gene, and determination of both nucleotide sequence and amino acid sequence of the tetanus toxin molecule have been achieved [see EMBO Journal, 5(10), 2495-2501, 1986 and Nucleic Acid Research, 14(19), 7809-7812, 1986 (the entire amino acid sequence of the whole tetanus toxin molecule is shown in SEQ ID NO. 1)]. Further, based on the above information on the entire nucleotide sequence and amino acid sequence, fragments of the tetanus toxin gene are expressed and synthetic peptides are produced as parts of the tetanus toxin molecule, and in addition, determination of the epitope regions of the tetanus toxin has been attempted using the expression products of the gene DNA fragments and the synthesized peptides [see Infection and Immunity, 57(11), 3498-3505, 1989 and Molecular Immunology, 31(15), 1141-1148, 1994]. However, tetanus vaccines comprising such tetanus toxin epitopes as active components have not been achieved.

SUMMARY OF THE INVENTION

The present inventor has long studied tetanus toxin to date for more than 20 years since the early 1970s, when purification of tetanus toxin to a high level could not be achieved and the detailed structure and properties of the tetanus toxin molecule were unknown. The present inventor extensively studied the toxin-producing ability of tetanus bacilli. He has further made extensive and intensive studies for developing a tetanus vaccine antigen which is extremely excellent with respect to diminution of adverse side effects of conventional tetanus vaccines comprising, as an antigen, the whole tetanus toxin toxoid, but also has an immunopotency which is substantially the same as that of the whole tetanus toxin toxoid. As a result, he found that a specific functional fragment antigen (hereinafter referred to simply as “FFA”) derived from tetanus toxin is effective as an antigen for a tetanus vaccine, and is extremely excellent with respect to diminution of adverse side effects. The present invention has been completed, based on the novel findings.

Therefore, it is an object of the present invention to provide a tetanus antigen which is extremely excellent with respect to diminution of adverse side effects of the conventional whole tetanus toxin toxoid, but also has an immunopotency which is substantially the same as that of a whole tetanus toxin toxoid.

It is another object of the present invention to provide a tetanus vaccine which is extremely excellent with respect to the diminution of adverse side effects of the current vaccines, and has an immunopotency which is substantially the same as that of the conventional whole tetanus toxoid vaccine.

A further object of the present invention is to provide a method for producing the above-mentioned tetanus vaccine.

Still a further object of the present invention is to provide a method for producing the above-mentioned functional fragment antigen (FFA) as a tetanus vaccine antigen.

The foregoing and other objects, features and advantages of the present invention will be apparent to those skilled in the art from the following detailed description and appended claims taken in connection with the accompanying sequence listing and drawings.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO. 1 is one form of the entire amino acid sequence of the whole tetanus toxin molecule used in the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

In the accompanying drawings:

FIG. 1(a) is a diagrammatic view of the structure of the whole tetanus toxin molecule used in the present invention;

FIG. 1(b) is a diagrammatic view of a nicked form of the whole tetanus toxin molecule;

FIG. 1(c) shows a tripartite [A-B·C] model of the whole tetanus toxin molecule;

FIG. 2(a) is a diagrammatic view showing a variety of the N-terminal amino acid sequences of the tetanus toxin functional fragment antigen (SEQ ID NO: 2-9; and

FIG. 2(b) is a diagrammatic view of a structural model of the whole tetanus toxin molecule.

DESCRIPTION OF REFERENCE CHARACTERS

S - - - S: disulfide bridge

*: nick

- - - : non-covalent bond

In the one-letter representation system for representing the amino acid residues of an amino acid sequence, the letters respectively represent the following amino acid residues:

A Alanine C Cysteine D Aspartic acid E Glutamic acid F Phenylalanine G Glycine H Histidine I Isoleucine K Lysine L Leucine M Methionine N Asparagine P Proline Q Glutamine R Arginine S Serine T Threonine V Valine W Tryptophan Y Tyrosine.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, there is provided a tetanus toxin functional fragment antigen, comprising at least one fragment which is substantially the same as that obtained by a process comprising the steps of splitting at least one peptide bond selected from peptide bonds individually connecting mutually adjacent amino acid residues in a partial amino acid sequence between two cysteine residues participating in forming a disulfide bridge present in the N-terminal of the entire amino acid sequence of a whole tetanus toxin molecule, splitting the disulfide bridge, and splitting non-covalent bonds [as indicated in FIG. 2(b)] between groups on the tetanus toxin molecule;

the tetanus toxin functional fragment antigen having:

(a) a molecular weight of from 90,000 to 110,000 as measured by an SDS-polyacrylamide gel electrophoresis method; and

(b) an isoelectric point of 7.25±0.5 as measured by an isoelectric focusing method.

For easy understanding of the present invention, the essential features and various preferred embodiments of the present invention are enumerated below.

1. A tetanus toxin functional fragment antigen, comprising at least one fragment which is substantially the same as that obtained by a process comprising the steps of splitting at least one peptide bond selected from peptide bonds individually connecting mutually adjacent amino acid residues in a partial amino acid sequence between two cysteine residues participating in forming a disulfide bridge present in the N-terminal of the entire amino acid sequence of a whole tetanus toxin molecule, splitting the disulfide bridge, and splitting non-covalent bonds between groups on the tetanus toxin molecule;

the tetanus toxin functional fragment antigen having:

(a) a molecular weight of from 90,000 to 110,000 as measured by an SDS-polyacrylamide gel electrophoresis method; and

(b) an isoelectric point of 7.25±0.5 as measured by an isoelectric focusing method.

2. The tetanus toxin functional fragment antigen according to item 1 above, wherein each of the at least one fragment independently has an amino acid sequence selected from the group consisting of SEQ ID NOS:10-17. The N-terminal amino acid sequence of each of SEQ ID NOS:10-17 is represented by SEQ ID NOS:2-9, respectively. SEQ ID NOS:2-9 are depicted below as amino acid sequences (1) to (8), respectively.

(1) KIIPPTNIRENLYNRTASLTDLGGELCIK (SEQ ID NO:2),

(2) IIPPTNIRENLYNRTASLTDLGGELCIK (SEQ ID NO:3),

(3) ENLYNRTASLTDLGGELCIK (SEQ ID NO:4),

(4) NLYNRTASLTDLGGELCIK (SEQ ID NO:5),

(5) NRTASLTDLGGELCIK (SEQ ID NO:6),

(6) TASLTDLGGELCIK (SEQ ID NO:7),

(7) SLTDLGGELCIK (SEQ ID NO:8), and

(8) GGELCIK (SEQ ID NO:9). has an amino acid sequence selected from the group consisting of SEQ ID NOS:2-9. The N-terminal amino acid sequence of each of SEQ ID NOS: 2-9 is depicted below as amino acid sequences (1) to (8), respectively.

3. The tetanus toxin functional fragment antigen according to item 1 or 2 above, which is stabilized with a fixative.

4. A tetanus vaccine comprising, as an active component, the tetanus toxin functional fragment antigen of any one of items 1 to 3 above in an effective immunogenic amount.

5. A combined vaccine comprising, as one of a plurality of active components, the tetanus toxin functional fragment antigen of any one of items 1 to 3 above in an effective immunogenic amount.

6. A method for producing a tetanus vaccine, which comprises stabilizing a tetanus toxin functional fragment antigen with a fixative,

the tetanus toxin functional fragment antigen comprising at least one fragment which is substantially the same as that obtained by a process comprising the steps of collecting and purifying an extracellular tetanus toxin from a culture filtrate of Clostridium tetani to obtain an extracellular tetanus toxin molecule, splitting a disulfide bridge present in the N-terminal of the entire amino acid sequence of the extracellular tetanus toxin molecule, and splitting non-covalent bonds between groups on the extracellular tetanus toxin molecule;

the tetanus toxin functional fragment antigen having:

(a) a molecular weight of from 90,000 to 110,000 as measured by an SDS-polyacrylamide gel electrophoresis method; and

(b) an isoelectric point of 7.25±0.5 as measured by an isoelectric focusing method.

7. A method for producing a tetanus toxin functional fragment antigen, comprising:

ligating a DNA coding for the tetanus toxin functional fragment antigen of item 1 or 2 above to a vector;

transforming host cells, exclusive of Clostridium tetani, with the vector; and

expressing the DNA coding for the tetanus toxin functional fragment antigen.

Hereinbelow, the present invention is described in detail.

In the present invention, Ala represents an alanine residue, Arg represents an arginine residue, Asn represents an asparagine residue, Asp represents an aspartic acid residue, Cys represents a cysteine residue, Gln represents a glutamine residue, Glu represents a glutamic acid residue, Gly represents a glycine residue, His represents a histidine residue, Ile represents an isoleucine residue, Leu represents a leucine residue, Lys represents a lysine residue, Met represents a methionine residue, Phe represents a phenylalanine residue, Pro represents a proline residue, Ser represents a serine residue, Thr represents a threonine residue, Trp represents a tryptophan residue, Tyr represents a tyrosine residue and Val represents a valine residue.

As mentioned above, the tetanus toxin functional fragment antigen of the present invention comprises at least one fragment which is substantially the same as that obtained by a process comprising the steps of splitting at least one peptide bond selected from peptide bonds individually connecting mutually adjacent amino acid residues in a partial amino acid sequence between two cysteine residues participating in forming a disulfide bridge present in the N-terminal of the entire amino acid sequence of a whole tetanus toxin molecule, splitting the disulfide bridge, and splitting non-covalent bonds [as indicated in FIG. 2(b)] between groups on the tetanus toxin molecule.

As a preferred embodiment of the tetanus toxin functional fragment antigen of the present invention, there can be mentioned the tetanus toxin functional fragment antigen, wherein each of the above-mentioned at least one fragment independently has an amino acid sequence selected from the group consisting of SEQ ID NOS:10-17. The N-terminal amino acid sequence of each of SEQ ID NOS:10-17 is represented by SEQ ID NOS:2-9, respectively. SEQ ID NOS:2-9 are depicted below as amino acid sequences (1) to (8), respectively.

(1) KIIPPTNIRENLYNRTASLTDLGGELCIK (SEQ ID NO:2),

(2) IIPPTNIRENLYNRTASLTDLGGELCIK (SEQ ID NO:3),

(3) ENLYNRTASLTDLGGELCIK (SEQ ID NO:4),

(4) NLYNRTASLTDLCGELCIK (SEQ ID NO:5),

(5) NRTASLTDLGGELCIK (SEQ ID NO:6),

(6) TASLTDLGGELCIK (SEQ ID NO:7),

(7) SLTDLGGELCIK (SEQ ID NO:8), and

(8) GGELCIK (SEQ ID NO:9).

Further, the tetanus toxin functional fragment antigen of the present invention may be stabilized with a fixative.

For further clarification of the essential features of the present invention, the technical features of the present invention will be described by explaining the development of the present invention.

Isolation of a Strain of Clostridium tetani having High Toxin-producing Ability:

In the present invention, a strain of Clostridium tetani having high toxin-producing ability is used. The present inventor isolated a substrain having high toxin-producing ability by single colony isolation from Harvard H47 strain, which is a known C. tetani strain derived from a known C. tetani strain called the Harvard strain [ATCC (American Type Culture Collection) accession No. 10779], and he designated the obtained substrain as “Clostridium tetani Harvard H47 strain Biken substrain” (hereinafter referred to simply as “Biken substrain”). Also, the present inventor found that production of tetanus toxin is under the control of genetic information carried by the plasmid DNA in the C. tetani cell (Biken Journal, 20, 105-115, 1977). Further, by using the culturing method based on the above finding, the present inventor succeeded in mass production of tetanus toxin by culturing the Biken substrain, and high purification of the tetanus toxin.

Thus, in the present invention, it is first necessary that a strain of Clostridium tetani having high toxin-producing ability is selected and used as a seed culture. As a seed culture, a culture of transformant of a microorganism, such as yeast, Escherichia coli, Bacillus subtilis or the like, which is obtained using the below-mentioned DNA coding for FFA, by genetic engineering techniques, can be used.

Mode of Formation of and Toxic Activity of Tetanus Toxin:

In the C. tetani cells, tetanus toxin is first produced in the form of a single polypeptide chain (whole tetanus toxin molecule in a non-nicked, intact form) having a molecular weight of about 150,000 (hereinafter, frequently referred to as “intracellular toxin”). Subsequently, by the autolysis of the cell, the tetanus toxin is released from the cells into the extracellular medium (hereinafter, the toxin released into the extracellular medium is frequently referred to as “extracellular toxin”). When the toxin is released from the cells, at least one bond in the peptide bonds connecting mutually adjacent amino acid residues in the partial amino acid sequence between two cysteine residues participating in forming the disulfide bridge present in the N-terminal of the whole amino acid sequence of the whole tetanus toxin molecule is split by a protease produced by C. tetani, to thereby form at least two polypeptide chains. However, the two polypeptide chains are united to each other by the disulfide bridge present in the N-terminal of the whole tetanus toxin molecule, that is, these polypeptide chains assume a nicked forms [see FIGS. 1(a) and 1(b); Biochemical and Biophysical Research Communications, 57, 1257-1262, 1974; ibid. 68, 668-674, 1976; ibid. 77, 268-274, 1977], and further, the two polypeptide chains are also united to each other by non-covalent bonds [see FIG. 2(b)]. Conversion of the tetanus toxin molecule from the intact form into the nicked form enhances the toxin activity of the tetanus toxin several times, and the nicking is essential for eliciting toxic action. Therefore, for saving labor in the preparation of the functional fragment antigen, it is preferred to use, as a starting material, the extracellular tetanus toxin, which has already been converted into the nicked form [see FIG. 1(b)].

Subunit Structure of Tetanus Toxin and the Mechanism of Manifestation of Toxicity of Tetanus Toxin:

As a result of the unique studies of the present inventor, two functionary complementary polypeptide chains, namely, L (light) chain and H (heavy) chain, were obtained from the whole tetanus toxin molecule. That is, the present inventor succeeded in isolating and purifying these fragments (L and H chains), to thereby obtain the L chain and H chain individually, which are native enough to be able to reproduce the toxin activity of the whole tetanus toxin molecule when these L and H chains are reconstituted into the whole tetanus toxin molecule, although each of the individually obtained L chain and H chain is not toxic.

In addition, the present inventor also successfully isolated and purified the following three fragments: the C-terminal half of the H chain (fragment C), a fragment (fragment A-B) obtained by removing fragment C from the whole tetanus toxin molecule, and a fragment (fragment B) obtained by separating fragment A (i.e., L chain) from the purified fragment A-B.

Further, after the preparation of the whole tetanus toxin molecule and the above-mentioned fragments A, B, C, A-B and B-C, i.e., all the three subunits of tetanus toxin and the complexes of the adjacent subunits, the present inventor examined the differences in functions of these fragments, and the relationship between the subunit structure of tetanus toxin and the mechanism of the toxic action of tetanus toxin, and proposed a “tripartite [A-B·C] molecular model” [see FIG. 1(c); Biken Journal, 26, 133-143, 1983; and “Botulinum Neurotoxin and Tetanus toxin”, edited by L.

L. Simpson, pp. 69-92 (Chapter 4), Academic Press, 1989].

This tripartite molecular model was accepted as the most appropriate molecular model of tetanus toxin at the 8th International Conference on Tetanus (1988). According to the tripartite [A-B·C] model, fragment C has the role of carrying the tetanus toxin molecule to the central nervous system (letter “C” means “Carrier”), fragment B has the role of binding the tetanus toxin molecule to the presynaptic membrane of the nerve cell and the role of transporting the tetanus toxin molecule into the cytoplasm (letter “B” means “Binding”), and fragment A has the role of exhibiting the toxic activity based on the enzyme activity (letter “A” means “Active”) (see “8th International Conference on Tetanus”, edited by G. Nistco et al. pp. 170-171, Phythagora Press, Rome-Milan, 1989; Infection and Immunity, 57, 3588-3593, 1989; Toxicon, 27, 385-392, 1989; ibid. 28, 737-741, 1990).

Variety of the Fragments of Tetanus Toxin:

Until 1989, there was no consensus on any of the length, molecular weight and nomenclature of each of the fragments of tetanus toxin among researchers in the world, and this situation gave difficulties in exchange of information and discussions on the structures function relationship of subunits of tetanus toxin among researchers. Therefore, it was desired to establish a common basis for the studies on tetanus toxin by using unitary definitions of fragment models.

In such a situation, the present inventor proposed the above-mentioned tripartite molecular model for the first time. That is, the present inventor pointed out the disadvantages of the absence of a consensus on the lengths, molecular weights and nomenclatures of the tetanus toxin fragments, and the present inventor emphasized the necessity of unitary definitions of fragments and proposed the above models [see the above-mentioned “Botulinum Neurotoxin and Tetanus toxin”, edited by L. L. Simpson, pp. 69-92 (Chapter 4)].

It is believed that the reason why a variety of fragments are obtained from the whole tetanus toxin molecule resides not only in the genetic differences of seed strains of C. tetani used by different researchers, but also in that there are delicate differences in various operation conditions employed by researchers for obtaining tetanus toxin and fragments thereof, such as the culturing conditions for the seed strain, the autolysis conditions for the cultured cells to obtain the extracellular toxin which has already been converted into a nicked form, and the treating conditions for an extracted intracellular toxin, that is, the conditions for digesting the extracted intracellular toxin by a protease into a nicked form, and the conditions for treating the extracted intracellular toxin with a reducing agent, a denaturing agent, a solubilizing agent or the like, wherein examples of conditions for treating the extracted intracellular toxin include the types of the enzyme and reagents, the treatment temperature, the treatment time, the concentration of the enzyme or reagent, the pH of the treating solution, and the physical conditions for the treatment of the extracted intracellular toxin, i.e., stirring or shaking, or keeping it in a stationary state.

Definition of “FFA” of the Present Invention:

The functional fragment antigen (FFA) of the present invention is a tetanus toxin functional fragment antigen, comprising at least one fragment which is substantially the same as that obtained by a process comprising the steps of splitting at least one peptide bond selected from peptide bonds individually connecting mutually adjacent amino acid residues in a partial amino acid sequence between two cysteine residues participating in forming a disulfide bridge present in the N-terminal of the entire amino acid sequence of a whole tetanus toxin molecule, splitting the disulfide bridge, and splitting non-covalent bonds [as indicated in FIG. 2(b)] between groups on the tetanus toxin molecule;

the tetanus toxin functional fragment antigen having:

(a) a molecular weight of from 90,000 to 110,000 as measured by an SDS-polyacrylamide gel electrophoresis method; and

(b) an isoelectric point of 7.25±0.5 as measured by an isoelectric focusing method.

As a result of the research by the present inventor, it has been found that various types of N-terminal amino acid sequences of FFA can be obtained [see FIG. 2(a)]. In the present invention, it is preferred that the tetanus toxin functional fragment antigen has an N-terminal amino acid sequence selected from the group consisting of the eight amino acid sequences shown in FIG. 2(a). Further, the tetanus toxin functional fragment antigen (FFA) of the present invention has an immunopotency which is substantially the same as that of the whole tetanus toxin toxoid. In addition, the tetanus toxin functional fragment antigen (FFA) of the present invention is extremely excellent with respect to the diminution of adverse side effects, as compared to conventional whole tetanus toxin toxoids. The term “immunopotency” means the ability to prevent the occurrence of the symptoms of tetanus. In the present invention, the term “having an immunopotency which is substantially the same as that of the toxoid of the whole tetanus toxin molecule” means that the FFA exhibits a relative potency (ratio of the potency of FFA, relative to the potency of the whole tetanus toxin toxoid) of 1±0.2, as measured by a method in which a vaccine containing FFA and a vaccine containing the whole tetanus toxin toxoid prepared by the method described in Reference Example 14 are subjected to measurement of immunopotency by the parallel line assay using the whole toxin toxoid of a known international unit of potency as a reference and using a challenge toxin of a known LD₅₀ (Median Lethol Dose) described in Example 1(5). The results are analyzed by the score method described in Reference Example 15.

Thus, the present invention also provides a single-antigen tetanus vaccine comprising FFA as an active component, such as a plain preparation, an adsorbed preparation or a lyophilized preparation, and a combined vaccine comprising FFA as one of a plurality of active components, such as a DPT combined vaccine, a DT combined vaccine, or a combined vaccine comprising FFA and at least one vaccine antigen selected from the group consisting of vaccine antigens other than FFA, such as Haemophilus influenzae b vaccine antigen, inactivated poliomyelitis vaccine antigen, inactivated hepatitis B vaccine antigen, inactivated Japanese encephalitis vaccine antigen and the like, and a method for producing the above-mentioned vaccines in large quantities.

(1) Seed Microorganisms:

With respect to a microorganism used as a seed culture for obtaining the functional fragment antigen (FFA) of the present invention, there is no particular limitation as long as the microorganism has high toxin-producing ability. Examples of such microorganisms include the substrain of Clostridium tetani Harvard strain, and other C. tetani strains having substantially the same or higher producing ability for tetanus toxin, as or than that of the Harvard strain. Specifically, for example, it is preferred to use the Biken substrain having high toxin-producing ability (Reference Example 1), obtained by single colony isolation from the Harvard H47 strain, which is a known C. tetani strain derived from the Harvard strain [deposited with ATCC (American Type Culture Collection) under the accession No. 10779].

Further, as a seed microorganism, there can also be used a transformant microorganism obtained by a method in which a microorganism, such as yeast, Escherichia coli, Bacillus subtilis or the like, is transformed with a gene coding for FFA, using genetic engineering techniques. Specifically, for example, as a seed microorganism there can be used Escherichia coli transformed with a large-quantity expression vector having a DNA encoding FFA operably ligated thereto, which transformed E. coli is obtained in accordance with the method described in Reference Example 2 mentioned below.

(2) Medium:

Conventional media can be used for culturing the seed microorganism for obtaining FFA. For example, a conventional liquid medium for culturing an anaerobic microorganism can be used for culturing the above-mentioned seed microorganisms. Examples of such liquid media include cooked meat medium, PYG (Peptone, Yeast extract, Glucose) medium, GAM (Gifu Anaerobic Medium) broth, Veal infusion medium, thioglycolate medium, liver-liver broth, RCM (Reinforced Clostridial Medium) broth and DRCM (Differential Reinforced Clostridial Medium) broth. If desired, in order to improve the growth characteristics of the microorganisms and/or the maintenance of the low oxidation-reduction potential, a medium can be modified by replacement, addition or removal of components thereof, or by changing the amount of components thereof. Among these media, liver-liver broth is preferred for use in preparing a seed culture, and a modified Latham medium designed by the present inventor (Reference Example 3) is preferred for use in producing tetanus toxin from the seed culture.

(3) Culture Conditions:

There is no particular limitation with respect to the conditions for culturing the seed microorganism for obtaining FFA. For example, a strain of C. tetani having high toxin-producing ability is cultured under culture conditions, in which the incubation temperature is from about 30 to about 37° C., preferably from about 34 to about 36° C., and the incubation time is from about 1 to about 8 days, particularly from about 1 to about 2 days for extracting the intracellular toxin, and particularly from about 4 to about 7 days for harvesting the extracellular toxin.

(4) Starting Materials for the Preparation of FFA:

In the present invention, FFA is prepared using the whole tetanus toxin molecules obtained from the cells cultured as described above. Examples of starting materials for the preparation of FFA include a cell extract (containing an intracellular tetanus toxin) of the microorganisms cultured as described above and a culture supernatant or a culture filtrate (each containing an extracellular tetanus toxin in a nicked form) obtained by a method in which the cultured cells are allowed to undergo autolysis, and the unlysed cells and cell debris contained in the resultant autolysis product are removed by centrifugation or filtration. When it is desired to prepare FFA from intracellular tetanus toxin, it is necessary to split the intracellular tetanus toxin with a protease, such as trypsin or chymotrypsin, thus converting the intracellular tetanus toxin into a nicked form. Therefore, for saving labor in the preparation process and achieving high yield, it is preferred to use, as a starting material, a culture supernatant or a culture filtrate each containing an extracellular tetanus toxin in a nicked form.

(5) Premiminary Purification of Tetanus Yoxin:

The whole tetanus toxin molecule in the starting material can be roughly purified by conventional methods. Examples of such conventional methods include salting out by using ammonium sulfate, alcohol precipitation, adsorption onto and desorption from a gel, and ultrafiltration by using commercially available membranes. In the present invention, the whole tetanus toxin molecule obtained by these preliminary purification methods is referred to as “partially purified whole tetanus toxin”.

(6) High Purification of Tetanus Toxin:

The partially purified whole tetanus toxin, obtained by the method described in item (5) above, can be highly purified by, for example, a method using both density-gradient ultracentrifugation and equilibrium density-gradient ultracentrifugation (see Unexamined Japanese Patent Application Laid-Open Specification No. 07-89951), or a method using an appropriate combination of conventional methods, such as ultracentrifugation, gel filtration, ion-exchange chromatography and high performance liquid chromatography. In the present invention, a highly purified whole tetanus toxin molecule obtained by these purification methods (hereinafter, frequently referred to simply as “highly purified tetanus toxin”) can be used as a material for preparation of FFA of the present invention. The highly purified tetanus toxin needs to be confirmed with respect to its eligibility for use as whole tetanus toxin molecules. The confirmation of the eligibility can be performed by determining, for example, MLD (Minimum Lethal Dose; Reference Example 4), Lf unit (Unit of flocculation; Reference Example 5), protein content (Reference Example 6), or the like.

(7) Preparation of FFA:

In the present invention, FFA is prepared from the above-mentioned highly purified tetanus toxin. When the highly purified tetanus toxin used for preparation of FFA is prepared from intracellular tetanus toxin, it is first necessary to digest mildly or split the intracellular tetanus toxin with a protease, such as trypsin or chymotrypsin, so as to convert the toxin into a nicked form. Two functionally complementary fragments of tetanus toxin can be prepared by a method comprising the steps of splitting a disulfide bridge present in the N-terminal of the purified tetanus toxin in the nicked form by a reducing agent, and splitting non-covalent bonds between groups on the tetanus toxin molecule by a denaturing agent. Examples of reducing agents include conventional reducing agents, such as sodium thioglycolate, dithiothreitol (hereinafter referred to simply as “DTT”), glutathione, mercaptoethanol, hydrogen sulfide, sodium borohydride, sodium sulfide, ammonium sulfide and the like. As denaturing agents, conventional denaturing agents can be used. Examples of these agents include guanidine thiocyanate, guanidine hydrochloride, urea, sodium dodecyl sulfate and the like. In the present invention, DTT and urea are preferred. With respect to a preferred manner of the use of DTT, for example, the final concentration of DTT in a solution containing the toxin protein in an amount of from about 1 to about 10 mg/ml is generally in the range of from 10 to 200 mM, preferably from 50 to 150 mM, and the reaction is conducted at 15 to 35° C. for 20 to 180 minutes. With respect to a preferred manner of the use of urea, for example, the final concentration of urea in a solution containing the toxin protein in an amount of from about 1 to about 10 mg/ml is generally in the range of from 0.5 to 10 M, preferably from 1 to 5 M, and the reaction is conducted at 5 to 35° C. for 10 seconds to 15 minutes. Each of these reagents is added to the starting material so that the concentration of the reagent falls within the above-mentioned respective concentration range. In the present invention, DTT is used in the form of a solution (see Reference Example 8; hereinafter, this solution is referred to as “DTT Tris buffer”), and the solution is added to the starting material in an amount about 5 to about 50 times by volume the amount of the starting material, to react DTT with tetanus toxin. Urea is used in the form of a saturated solution or directly in the form of crystals.

As a result of the above-mentioned treatments with DTT and urea, the whole tetanus toxin molecule in the nicked form is split to obtain a solution containing FFA. By diluting the solution containing FFA to thereby lower the concentration of urea, FFA can be obtained as a highly purified tetanus toxin fragment by fractionation or separation using an absorbance at 280 nm as an index (see Reference Example 7). The purification can be performed by, for example, a combined method using both density-gradient ultracentrifugation and equilibrium density-gradient ultracentrifugation (see Unexamined Japanese Patent Application No. 07-89951), SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis), gel filtration, membrane filtration, ion-exchange chromatography, high performance liquid chromatography and the like. By these purification methods, two fractions of different molecular weights, corresponding to two peaks appearing at 280 nm, are obtained, and FFA is found in the fraction of the larger molecular weight. This fraction containing FFA is used as an active component for an FFA tetanus vaccine. The whole tetanus toxin molecule can be prepared according to the method described below (Reference Example 13). The molecular weight, antigenic specificity, amino acid sequence and the like of each of the whole tetanus toxin molecule and FFA can be determined by, for example, SDS-PAGE (Reference Example 10), a precipitation reaction in gel (Reference Example 11) and a method using a peptide sequencer (Reference Example 12).

(8) Stabilization of FFA:

In the FFA of the present invention, a whole or most of the fragment A region, which is the active site exhibiting the toxicity, is absent, so that the FFA of the present invention has no toxicity. Therefore, the FFA as such can be used as a toxoid without detoxification. However, for stabilizing the stereochemical structure of FFA as an antigen, it is preferred to perform a stabilization (fixation) of the tetanus toxin FFA. Examples of fixatives (fixing or stabilizing reagent) include conventional detoxifying agents, such as formalin, glutaraldehyde, β-propiolactone and the like. For example, when formalin is used as a fixative, it is preferred that the volume ratio of formalin to the FFA solution is approximately within the range of from 0.0004 to 0.7% (v/v). The fixation temperature is approximately within the range of from 3 to 37° C. and the fixation time is approximately within the range of from 5 to 180 days. When there is a danger that the antigenecity of FFA is deteriorated by the fixation, the fixation conditions are rendered moderate by lowering the concentration of the fixative, lowering the fixation temperature or adding neutral amino acids, such as glycine and serine, or basic amino acids, such as lysine and arginine. When free formaldehyde remains in the solution after the fixation, if desired, it can be neutralized by addition of sodium hydrogensulfite in an equivalent amount to the amount of the formaldehyde, or can be removed by filtration using a membrane or by dialysis. After the fixation treatment, the FFA solution is stored at 4° C. for subsequent use as a bulk solution of tetanus toxin vaccine for preparing a tetanus vaccine. After the fixation treatment, the treated whole tetanus toxin molecule and treated FFA are referred to as a “whole tetanus toxin toxoid” and an “FFA toxoid”, respectively.

(9) Preparation of FFA Tetanus Vaccine:

The FFA toxoid bulk solution obtained in item (8) above can be diluted so as to obtain a vaccine comprising the FFA toxoid in an effective immunogenic amount. For example, the bulk solution can be diluted with an isotonic solution of salts, or with buffer or a medium for tissue culture so that the protein content of the vaccine becomes from 20 to 200 μg for a single-antigen toxoid, or 8 to 80 μg for an adsorbed toxoid.

When the bulk solution is diluted as mentioned above, a stabilizer for increasing the heat resistance of the vaccine or an adjuvant as a supplementary agent for increasing the immunopotency can be added to the vaccine solution. It is preferred that the stabilizer is added to the vaccine in an amount of from 0.01 to 10 % (w/v), and the adjuvant is added in an amount of from 0.1 to 50 mg per ml of the vaccine. Examples of stabilizers include saccharides, amino acids, gelatin hydrolyzate, human albumin and the like. Examples of adjuvants include gels capable of effecting sustained release of antigens, such as calcium phosphate, aluminum phosphate, aluminum sulfate, alumina, bentonite and the like; and antibody production inducing agents, such as muramyldipeptide derivatives, oils and the like.

Subsequently, the single-antigen vaccine or adsorbed vaccine is dispensed into small containers, such as ampuls or vials. Then, the containers are sealed hermetically, for example, by fusion sealing, and the sealed vaccine is provided as a plain or adsorbed preparation. When the vaccine is lyophilized after dispensation, the vaccine can be provided as a lyophilized preparation. Further, the vaccine can be provided in the form of a combined vaccine preparation, such as a DT binary vaccine, a DPT ternary vaccine, a quaternary vaccine or the like.

Each of these preparations is subjected to various tests to verify the eligibility for use as a toxoid or a vaccine. That is, each of these preparations is subjected to various tests on effectiveness, safety and homogeneity in accordance with a relevant provision (such as a provision entitled “tetanus toxoid”, a provision entitled “adsorbed tetanus toxoid”, a provision entitled “diphtheria-tetanus combined toxoid”, or a provision entitled “diphtheria-pertussis-tetanus combined vaccine”) in the Notification No. 217 of the Japanese Ministry of Health and Welfare: “Seibutsugakuteki Seizai Kijun (Minimum Requirements for Biological Products), to thereby verify its eligibility for use as a vaccine. Only the verified preparations are put to practical use. With respect to the manner of administration, for example, a preparation is usually administrated by subcutaneous or intramuscular injection in an amount of 0.5 ml per person. In the case of a lyophilized preparation, before use, it is dissolved in sterile distilled water so as to obtain a solution having the original volume before lyophilization.

(10) Measurement of Immunopotency of FFA Tetanus Vaccine:

The immunopotency of FFA tetanus vaccine of the present invention is measured using immunized animals (guinea pigs or mice) by a potency assay using a challenge toxin of a known LD₅₀ (Median Lethal dose), or an assay of toxin-neutralizing activity of antisera, in accordance with the provision entitled “tetanus toxoid” or the provision entitled “adsorbed tetanus toxoid” in the Notification No. 217 of the Japanese Ministry of Health and Welfare: “Seibutsugakuteki Seizai Kijun (Minimum Requirements for Biological Products)”. In addition to the above-mentioned tests, in the present invention, the relative immunopotency of FFA to that of the whole tetanus toxoid is measured by performing a further potency assay using a challenge toxin, wherein the score method (Reference Example 15) is employed.

(11) Animal Tests on the Adverse Side Effects of FFA Tetanus Toxoid:

Animal tests on the side effects of the FFA tetanus vaccine of the present invention can be conducted in accordance with conventional methods. Examples of animal tests which can be employed for evaluating the adverse side effects of tetanus toxoid include the passive cutaneous anaphylaxis (PCA) test using rats which is based on an immediate type allergic reaction, the footpad reaction test using mice which is based on a delayed type allergic reaction, and the intradermal reaction test using guinea pigs (Reference Example 16) which test is based on a delayed type allergic reaction.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinbelow, the present invention will be described in more detail with reference to the following Examples and Reference Examples, but they should not be construed as limiting the scope of the present invention.

In the following Reference Examples, the guidelines for carrying out the present invention are specifically shown.

REFERENCE EXAMPLE 1

Isolation of a Single Colony of a C. tetani Strain having High Toxin-producing Ability:

Colonies of C. tetani Harvard A47 strain are formed on a plate of Zeissler's blood agar [prepared by adding glucose and defibrinated bovine blood to a commercially available non-modified agar medium in such amounts as would give a final glucose concentration of 2% (w/v) and a final defibrinated bovine blood concentration of 20% (v/v), followed by mixing], and the formed colonies are individually inoculated into a modified Latham medium (see Reference Example 3 below) and incubated to obtain cultures. With respect to each of the obtained cultures, the Lf value is measured in accordance with the method as described in Reference Example 5 below. From the above-mentioned colonies, the colony used for the culture having the highest Lf value is used as a Biken substrain of the C. tetani Harvard A47 strain, which has high toxin-producing ability.

REFERENCE EXAMPLE 2

Preparation of Transformants having High Toxin-producing Ability for FFA:

A DNA of tetanus toxin gene [see EMBO JOURNAL, 5(10), 2495-2502, 1986 and Nucleic Acid Research 14(19), 7809-7812, 1986] is digested with the restriction enzymes, thereby obtaining a 2.7 kb DNA fragment (Stu I-Bsp HI) coding for FFA. The obtained DNA fragment is inserted into and ligated to pSN508 [which is a vector capable of a large-quantity expression in E. coli (see U.S. Pat. No. 4,703,005)] to obtain a recombinant expression vector. Then, the obtained recombinant expression vector is introduced into E. colistrain CSH26 to form transformants having high toxin-producing ability for FFA. When the production of the FFA is conducted by culturing the above-mentioned transformants, it is not necessary to perform the treatments described below, such as a protease digestion of the whole tetanus toxin molecule, and a treatment using dithiothreitol or urea, but purification is necessary.

REFERENCE EXAMPLE 3

Composition of the Modified Latham Medium (per 1 liter of the medium):

Polypeptone 20 g Bovine heart extract 10 g Glucose 8.0 g Sodium chloride 2.5 g Magnesium sulfate (heptahydrate) 0.1 g Cystine 0.125 μg Calcium pantothenate 1.0 mg Uracil 1.25 mg Nicotinic acid 0.25 mg Thiamine 0.25 mg Riboflavin 0.25 mg Pyridoxine 0.25 mg Biotin 2.5 μg Vitamin B₁₂ 0.05 μg Folic acid 100 μg Iron(III) chloride (hexahydrate) 32 mg Iron sulfate (heptahydrate) 0.2 g (The pH was adjusted to 7.0 using 7N NaOH)

REFERENCE EXAMPLE 4

MLD (Minimum Lethal Dose):

0.1 to 0.5 ml of each of dilutions of the tetanus toxin-containing solution which have been prepared by successively diluting tetanus toxin at logarithmic intervals of 10^(0.5) is individually injected subcutaneously or intramuscularly to OF1 mice (weighing 20 to 25 g) at the of the thigh of the left hind leg. MLD is determined, based on the dose (log. of dose)-response (time to death) curve [see “Proceedings of the 6th International Conference on Tetanus (Lyon, 1981)” pp. 21-32].

REFERENCE EXAMPLE 5

Lf unit (Unit of Flocculation):

The Lf value of a toxin solution can be measured by the Ramon's method. (Biken Journal, 7, 137-152, 1964). 1 Lf of the toxin, which is the amount of the toxin which reacts with 1 unit of the antitoxin, is measured. Also, the measurement of the above-mentioned Lf value can be conducted using SRID (Single Radial Immunodiffusion) (see Immunochemistry, 2, 235-254, 1965).

REFERENCE EXAMPLE 6

Measurement of Protein Content:

The protein content is measured in accordance with the “modified method of Lowry et al.” in which the color reaction of a protein and a phenol reagent is evaluated by colorimetry. Hereinafter, this method is simply referred to as a “phenol reagent method”.

REFERENCE EXAMPLE 7

Identification of Protein Fractions and Comparison on Protein Concentrations Between the Protein Fractions:

The identification of protein fractions and the comparison on protein concentrations between the protein fractions are conducted by measuring the absorbance of ultraviolet rays having a wave length of 280 nm (hereinafter, referred to as an “absorbance at 280 nm”) of a sample.

REFERENCE EXAMPLE 8

Preparation of a DTT-Tris Buffer (100 mM):

A DTT-Tris buffer is prepared by mixing 50 mM tris(hydroxymethyl)aminomethane-HCl (hereinafter, simply referred to as “Tris”), 1 mM ethylenediamine-tetraacetate-4 Na (hereinafter, simply referred to as “EDTA”) and 100 mM DTT. The pH of the DTT-Tris buffer is adjusted to 8.2 using 1/10 M HCl.

REFERENCE EXAMPLE 9

Preparation of a Phosphate Buffer:

A phosphate buffer is prepared by mixing equal molar amounts of disodium monohydrogenphosphate and potassium dihydrogenphosphate solutions. The amounts of the solutions to be mixed are appropriately chosen so that the resultant phosphate buffer has a desired pH value.

REFERENCE EXAMPLE 10

Measurement of the Molecular Weights of Proteins by Using SDS-PAGE:

In the SDS-PAGE, an SDS-PAGE gel having a gel content of 7.5% (w/v), 7.0% (w/v) (containing 2M urea), 5.0% (w/v) or the like can be used. As a buffer solution, for example, 10 mM Tris-77 mM glycine buffer (pH 8.6) can be used. After the electrophoresis, the gel is stained using Coomassie brilliant blue. The molecular weight of each of the proteins is individually determined from the ratio of the migration distance of the sample protein to the migration distance of the marker dye or the protein having known molecular weight. As a result of the measurements conducted in accordance with the above method, it is found that the molecular weights (×10⁴) of the FFA and the whole tetanus toxin molecule are 100,000 and 150,000, respectively.

REFERENCE EXAMPLE 11

Determination of the Antigenic Specificity by Using Double Immunodifusion:

The antigenic specificity is determined by the method of Ouchterlony using 1% (w/v) agarose in 50 mM Tris-0.6 M Glycine buffer (containing 1 mM EDTA; pH 8.5) and horse anti-tetanus toxin serum from which nonspecific antibodies are removed. A cross-reaction of antigenicity is observed between the FFA and the whole tetanus toxin molecule.

REFERENCE EXAMPLE 12

Determination of the Amino Acid Sequence of the FFA:

The determination of the amino acid sequence of the FFA is conducted using an automatic amino acid sequencer, such as Applied Biosystem Procise Type 492 manufactured and sold by Perkin Elmer, U.S.A. The FFA obtained in Example 1 below has N-terminal amino acid sequence (7) of FIG. 2(a), and the FFA obtained in Example 7 below has N-terminal amino acid sequence (4) of FIG. 2(a). Further, by appropriately choosing the conditions for cultivation of C. tetani, an FFA having N-terminal amino acid sequence (8) of FIG. 2(a) can be obtained. FFA's respectively having N-terminal amino acid sequences (1) to (3) and (6) of FIG. 2(a) can be prepared from the intracellular tetanus toxin by appropriately choosing the conditions for digesting the intracellular tetanus toxin molecule with trypsin to convert the toxin molecule to a nicked form thereof, such as an enzyme concentration, a reaction time and a reaction temperature. An FFA preparation having N-terminal amino acid sequence (5) of FIG. 2(a) can be prepared by digesting the intracellular tetanus toxin molecule with chymotrypsin to convert the toxin molecule to a nicked form thereof. Thus, in the present invention, 8 different types of FFA's respectively having N-terminal amino acid sequences (1) to (8) of FIG. 2(a) can be obtained. These 8 types of FFA's may be used as an active component of a tetanus vaccine individually or in combination.

REFERENCE EXAMPLE 13

Determination of the Isoelectric Point of FFA:

The isoelectric point of FFA is determined by an isoelectric focusing method using a commercially available gel, for example, a gel manufactured and sold under the tradename “Phast Gel IEP 3-9” by Pharmacia Biotech, Sweden. As a result of the measurements conducted in the Examples in accordance with the above method, it was found that the FFA of the present invention has an isoelectric point within the range of 7.25±0.5.

REFERENCE EXAMPLE 14

Preparation of the Whole Tetanus Toxin Molecule:

The seed culture of the C. tetani strain obtained in Reference Example 1 is inoculated into a culture medium described in Reference Example 3 for 44 hours and the bacterial cells are harvested by centrifugation at 10,000×g at 4° C. for 25 minutes. 1 M NaCl solution containing 0.1 M sodium citrate is added in an amount of {fraction (1/30)} volume of the cell culture to extract the intracellular toxin by gentle agitation. The resultant mixture is stirred at 4° C. overnight to extract tetanus toxin, and then is centrifuged at 10,000×g at 4° C. for 30 minutes to remove cells and cell debris. Using the resultant supernatant as a starting material, the toxin is purified in substantially the same manner as in Example 1 below to obtain a purified whole tetanus toxin preparation.

REFERENCE EXAMPLE 15

Score Method for Evaluating Immunopotency:

Relative potency of tetanus toxoid is evaluated using a score method. Illustratively stated, animals for immunological experiment, which have been injected with tetanus vaccine, are challenged with toxin, and observed over a week for the symptoms of tetanus. The severity of the symptoms was evaluated in accordance with the following criteria by the score method:

Results Score The animal dies on the 1st day: 0 The animal dies on the 2nd day: 1 The animal dies on the 3rd day: 2 The animal dies or exhibits serious 3 symptoms (such as a tonic convulsion, a difficulty in walking and a respiratory distress) between the 4th and 7th day: The animal survives through one week 4 with slight symptoms [such as a local paralysis of the abdominal muscle on a side opposite to the side on which the injection had been made (this symptom is checked by observing the animal hung by its tail)]: The animal survives through one week 8 without any symptom:

The obtained scores are analyzed by a computer using a software designed for statistical analysis for the parallel-line assay, in which the correlation analysis of the scores is conducted in terms of uniformity, linearity and parallelity to evaluate the relative immunopotency of the tetanus toxoid to that of the standard tetanus whole toxin toxoid of a known international units.

REFERENCE EXAMPLE 16

Intradermal Reaction Using Guinea Pigs:

Three groups of guinea pigs are sensitized by intramuscular injection with 1 ml of tetanus vaccine (10 μg protein/ml) per 1 guinea pig, wherein the above-mentioned three groups of guinea pigs are sensitized with (1) commercially available partially purified whole toxin toxoid, (2) purified whole toxin toxoid and (3) FFA vaccine, respectively. After 4 weeks from the sensitization, the back of each of the guinea pigs is shaved and then, the guinea pigs are individually injected intradermally with 0.1 ml of the above-mentioned tetanus toxoid onto the back thereof. The protein concentration of the tetanus toxin solution is varied (32.0, 10.0 and 3.2 μg) depending on the group to which a guinea pig belongs. 24 hours after the injection, with respect to each of the guinea pigs, the portion which has been injected is examined to see whether or not a skin reaction, an erythema, an induration and/or a necrosis occurs. When the occurrence of an erythema is observed, the diameter of the erythema is measured.

EXAMPLE 1

(1) Production and Purification of the Whole Tetanus Toxin Molecule (nicked form)

Into each of 20 tubes (diameter: 6 cm; height: 38 cm) containing 450 ml of a modified Latham medium (see Reference Example 3), 5 ml of the seed culture of a Biken substrain of the C. tetani Harvard A47 strain (see Reference Example 1) was inoculated. The tubes with loose cotton plugs were incubated at 35° C. for 5 days until the cultures became clear. These cultures were centrifuged at 10,000×g at 4° C. for 30 minutes to obtain clean culture supernatants. 8.5 liters of the culture supernatants thus obtained as starting materials were filtered and used for purification of whole tetanus toxin molecules (nicked form).

A saturated solution (at 25° C.) of ammonium sulfate (pH 7.0) was added to and mixed with the starting material in an ice-water bath to salt out a fraction having an ammonium sulfate saturation of 20% to 40%. The obtained fraction was suspended in 65 ml of 0.06 M phosphate buffer (pH 7.5, 4° C.) (see Reference Example 9) to obtain a suspension having an ammonium sulfate saturation of 40%. The obtained suspension was subjected to centrifugation at 15,000×g at 4° C. for 30 minutes to obtain a precipitate. The obtained precipitate was washed by resuspending the precipitate in 22 ml of the above-mentioned phosphate buffer, and the resultant was subjected to centrifugation under the same centrifugation conditions as mentioned above. The washed precipitate was dissolved in 22 ml of 0.1 M phosphate buffer (pH 7.5, 4° C.) and subjected to ultracentrifugation at 100,000×g at 4° C. for 2 hours to remove the precipitated residue, thereby obtaining 20 ml of a supernatant. The obtained supernatant was filtered through Acrodisc membrane (pore diameter: 0.2 μm) (manufactured and sold by Gelman Co. Ltd., Germany), and the resultant filtrate of partially purified toxin was subjected to gel filtration by using Ultrogel AcA 34 column (inner diameter: 2.5 cm, length: 100 cm) (manufactured and sold by LKB-Pharmacia, Sweden) equilibrated with 0.1 M phosphate buffer (pH 7.5). In the gel filtration, elution was carried out at 4° C. using the above-mentioned phosphate buffer (flow rate: 9 ml/hr), and the resultant eluate was fractioned into 1 ml fractions. With respect to each of the fractions, an absorbance at 280 nm (see Reference Example 7) was measured. The fractions having a high absorbance were pooled, thereby obtaining a gel filtration eluate in a total volume of 5 ml.

With respect to the obtained eluate, the measurement of the protein content in accordance with the phenol reagent method (see Reference Example 6), the measurement of MLD per 1 mg of protein (see Reference Example 4) and the measurement of Lf per 1 mg of protein (see Reference Example 5) were conducted. As a result, the protein content of the eluate was 60 mg/ml, Lf was 400 and MLD was 3.5×10⁷.

Further, 0.2 ml of the eluate was subjected to high performance liquid chromatography (HPLC) using TSK G3000 SW column (inner diameter: 0.75 cm, length: 60 cm) (manufactured and sold by Tosoh Corp., Japan) equilibrated with 0.1 M phosphate buffer (pH 6.8), in which elution was carried out using the above-mentioned phosphate buffer (flow rate: 0.6 ml/minute) to thereby obtain fractions. The absorbance at 280 nm of each of the fractions was measured. As a result, a single sharp peak was observed, which indicates that the whole tetanus toxin molecule was highly purified. In the preparation of FFA described below, the eluate obtained above was used as a solution of a highly purified entire length tetanus toxin molecule (nicked form) (i.e., a highly purified tetanus toxin solution).

(2) Preparation of FFA

18 ml of a DTT-Tris buffer (see Reference Example 8) was added to 2 ml of the highly purified tetanus toxin solution, followed by mixing. The resultant mixture was reacted at 25° C. for 60 minutes to reduce the disulfide bridge present in the whole tetanus toxin molecule. The resultant mixture was treated with urea by adding 4.8 g of a solid urea to the mixture, followed by mixing to dissolve the solid urea in the mixture (final urea concentration: 4 M). To the resultant was added 20 ml of 50 mM Tris buffer (containing 0.6 M glycine, 1 mM EDTA and 1 mM DTT; pH 8.5) and the resultant mixture was condensed using Amicon Ultrafiltration System (manufactured and sold by Grace Company, U.S.A.),” thereby obtaining 3 ml of a condensate. The obtained condensate was subjected to gel filtration by using Ultrogel AcA 44 column (inner diameter: 1.5 cm, height: 90 cm) equilibrated with 50 mM Tris buffer (containing 0.6 M glycine, 1 mM EDTA and 1 mM DTT and 2 M urea; pH 8.5). In the gel filtration, elution was carried out using the above-mentioned 50 mM Tris buffer (flow rate: 5 ml/hr) and the resultant eluate was fractioned into 1.2 ml fractions. With respect to each of the fractions, the absorbance at 280 nm was measured. As a result, two peaks (peak 1 and peak 2) were observed. From the obtained fractions including two fractions each exhibiting a peak at 280 nm (i.e., a peak 1 fraction and a peak 2 fraction, wherein the peak 1 fraction was obtained earlier than the peak 2 fraction), a set of 5 fractions (total amount: 6 ml) successively obtained starting from the peak 1 fraction and another set of 5 fractions (total amount: 6 ml) successively obtained starting from the peak 2 fraction were collected to obtain a collected fraction 1 and a collected fraction 2, respectively.

Each of the highly purified tetanus toxin solution, the collected fraction 1 and the collected fraction 2 was subjected to SDS-PAGE (see Reference Example 10) for determination of the approximate molecular weight and to gel precipitation reaction (see Reference Example 11) for determination of the antigenicity. As a result, the molecular weights of the purified tetanus toxin, the collected fraction 1 and the collected fraction 2 were approximately 150,000, 100,000 and 50,000, respectively. With respect to the antigenicity, the cross-reaction of the antigenicity was observed between the highly purified tetanus toxin solution and each of the collected fraction 1 and the collected fraction 2, whereas no cross-reaction was observed between the collected fraction 1 fraction and the collected fraction 2, which indicates that these two types of collected fractions had completely different antigenicities. Further, the protein contents of the collected fraction 1 and the collected fraction 2 were determined by the phenol reagent method. The protein contents of the collected fraction 1 and the collected fraction 2 were 15 mg/ml and 8 mg/ml, respectively. From the above results, it was confirmed that the collected fraction 1 contains FFA. In the operations described below, the collected fraction 1 was used as an FFA solution.

(3) Stabilization of the FFA

4 ml of the FFA solution was dialyzed against 1/15 M phosphate buffer (pH 7.8) at 4° C. overnight. After completion of the dialysis, the above-mentioned phosphate buffer was added to and mixed with the dialyzed FFA solution to thereby obtain a solution having a total volume of 380 ml. 20 ml of 500 mM lysine solution was added to the above-mentioned FFA solution so that the protein content of the FFA solution became 600 μg/ml. To the resultant FFA solution was added formalin in an amount such that the final formalin concentration became 0.2% (v/v), and the resultant mixture was subjected to incubation at 37° C. for 14 days to stabilize the FFA. Then, the stabilized FFA was dialyzed against a 0.85% (w/v) NaCl solution at 4° C. overnight to remove formaldehyde and lysine, and the dialyzate was filtered through Acrodisc membrane (pore diameter: 0.22 μm) for sterilization, thereby obtaining 380 ml of a filtrate. The obtained filtrate was stored at 4° C., and was used as a bulk FFA tetanus vaccine solution described below.

(4) Preparation of a Sample FFA Tetanus Vaccine

The bulk FFA tetanus vaccine solution was diluted with a 0.85% (w/v) NaCl solution so as to obtain a diluted solution having a final protein concentration of 50 μg/ml. An equivolume of Al(OH)₃ gel suspension [Al(OH)₃ content: 2 mg/ml] was added to the obtained diluted solution, followed by mixing. The resultant mixture was allowed to stand at 4° C. overnight so that the FFA was adsorbed on the Al(OH)₃ gel, thereby obtaining a sample vaccine. With respect to the obtained sample vaccine, the activities thereof (immunopotency) and the safety thereof (toxicity or adverse side effects) were evaluated as described below.

(5) Evaluation of the Immunopotency of the Sample FFA Tetanus Vaccine

The evaluation of the immunopotency of the sample FFA tetanus vaccine was conducted by experiments using mice. As a control, another vaccine was prepared in substantially the same manner as in the items (3) and (4) above, except that the solution of the whole tetanus toxin molecule prepared in Reference Example 14 was used. With respect to each of the sample vaccine and the control vaccine, serial 2.5-fold dilution with a 0.85% (w/v) NaCl solution was conducted to obtain dilutions having different dilution ratios, which were administered to mice as described below, wherein at least three dilutions had dilution ratios such that the dilutions exhibited dose-response relationships falling within the linear region of the dose-response curve. Using the obtained vaccine dilutions (i.e., sample vaccine dilutions and control vaccine dilutions), the immunization of mice was conducted as follows. The sample vaccine dilutions (each having the amount of 0.5 ml) were, respectively, administered to 10 randomly selected ddy/s female mice (each weighing 22 to 26 g) by subcutaneous injection to the inside of the thigh of the left hind leg. Four weeks after the injection, each of the immunized mice was challenged with 100 LD₅₀ standard toxin (Lot TA-4B) (provided by the National Institute of Health of Japan) by subcutaneous injection. The above operations were also conducted using the control vaccine dilutions instead of the sample vaccine dilutions. After the above operations, observations were made over a week as to whether or not the mice were alive, and as to the symptoms of the mice which were alive. Results of the observations were evaluated by the score method (see Reference Example 15). The obtained scores were analyzed with respect to variance and correlation by a computer using a software for the statistical analysis. From the results of the statistical analysis, the relative immunopotency of the sample FFA tetanus vaccine (the ratio of the immunopotency of the sample vaccine relative to the immunopotency of the vaccine comprising the whole tetanus toxin toxoid, wherein the immunopotency of the control vaccine is defined as 1.0) was calculated. The above experiment was repeated four times, and the obtained values of the relative immunopotency were statistically analyzed by a computer. Results are shown in Table 1. The immunopotency of the sample FFA tetanus vaccine was substantially the same as that of the control vaccine comprising the whole tetanus toxin toxoid.

(6) Experiments Using Guinea Pigs to Evaluate the Degree of Adverse Side Reactions Caused by the Intradermal Reaction of the Sample FFA Tetanus Vaccine.

The intradermal reactions were conducted using guinea pigs (std. Hartley, weighing 300 to 350 g, 5 weeks old, female) (obtained from Japan SLC, Inc., Japan) in accordance with the method described in Reference Example 16. The sensitization of the guinea pigs was conducted as follows. As antigens for sensitizing the guinea pigs, use was made of the sample FFA tetanus vaccine (FFA), a commercially available tetanus toxoid presumably containing a whole tetanus toxin toxoid (conventional toxoid) and the vaccine comprising the purified whole tetanus toxin toxoid (whole toxin toxoid). Each of these antigens was diluted using a 0.85% (w/v) NaCl solution so that the final protein concentration and the final Al(OH)₃ concentration became 10 μg/ml and 0.2 mg/ml, respectively, to thereby obtain three types of antigen dilutions. The guinea pigs were randomly divided into nine groups each consisting of three guinea pigs, and the above-obtained three types of the antigen dilutions were, respectively, administered to three guinea pigs of each of the nine groups. After completion of the sensitization period (4 weeks), the sensitized guinea pigs were challenged by the above-mentioned antigens by the following method. With respect to each of the above-mentioned antigens, three types of dilutions thereof respectively having final protein concentrations of 3.2, 1.0 and 0.32 μg/ml were prepared using a 0.85% (w/v) NaCl solution. The resultant nine types of dilutions (consisting of three types of dilutions of the FFA, three types of dilutions of the conventional toxoid and three types of dilutions of the whole toxin toxoid) were administered to the guinea pigs so that the guinea pigs belonging to the same group took the administration of the same type of dilution, wherein the dose of each of the dilutions was 0.1 ml. As a control, 0.1 ml of a 0.85% (w/v) NaCl solution was intradermally injected to each of three guinea pigs which had respectively been sensitized with the above-mentioned antigens in substantially the same manner as mentioned above. The results are shown in Table 2. With respect to each of the guinea pigs which had taken the administration of the sample FFA tetanus vaccine, the occurrence of the intradermal reaction was either undetectable or markedly slight as compared to that of the guinea pigs which had taken the administration of the conventional vaccine and the guinea pigs which had taken the administration of the vaccine comprising the whole tetanus toxin toxoid.

EXAMPLE 2

(1) Preparation of the Bulk FFA Tetanus Vaccine Solution

0.5 Liter of a seed culture of a Biken substrain of C. tetani Harvard A47 strain was inoculated in 80 liters of the modified Latham medium contained in a stainless steel tank having a volume of 100 liters (diameter: 60 cm, height: 50 cm). The tank was sealed by means of a silicone sheet, and the seed culture was incubated at 35° C. for 6 days to thereby obtain a culture. The obtained culture was filtered through a celite-filter paper for sterilization, thereby obtaining 75 liters of a filtrate. The obtained filtrate was concentrated using the “pericon cassette system” (manufactured and sold by Millipore, U.S.A.), thereby obtaining 7 liters of a condensate. Using the obtained condensate as a starting material, the purification of the whole tetanus toxin molecule, the preparation of the FFA and the stabilization of the FFA were performed in substantially the same manner as in Example 1, thereby obtaining 450 ml of a bulk FFA tetanus vaccine solution having a protein concentration of 600 μg/ ml.

(2) Production of an FFA Tetanus Plain vaccine Preparation

The bulk FFA tetanus vaccine solution obtained above was diluted using 1/75 M phosphate buffer (pH 6.5) so that the final protein concentration of the vaccine preparation would become 60 μg/ml. To the resultant dilution were individually added sucrose, L-arginine and Haemaccel (manufactured and sold by Hoechst Aktiengesellschaft, Germany) in this order in amounts such that the final concentrations of sucrose, L-arginine and Haemaccel became 3% (w/v), 1% (w/v) and 2% (w/v), respectively, followed by mixing to obtain a single-antigen vaccine preparation. The obtained preparation was dispensed in glass vials each having a volume of 1 ml, so that each vial contained 0.6 ml of the preparation, and then, the vials were sealed. The obtained single-antigen vaccine preparation was subjected to various tests in accordance with a provision entitled “tetanus toxoid” in the Notification No. 217 of the Japanese Ministry of Health and Welfare: “Seibutsugakuteki Seizai Kijun (Minimum Requirements for Biological Products)”. As a result, the obtained preparation was verified as a qualified vaccine.

EXAMPLE 3

Production of an Adsorbed FFA Tetanus Vaccine Preparation

The bulk FFA tetanus vaccine solution obtained in Example 2 was diluted using 1/40 M phosphate buffer (pH 6.0) so that the final protein concentration of the vaccine preparation became 60 μg/ml. To the resultant dilution was added an aluminum phosphate gel in an amount such that the final aluminum phosphate gel concentration of the vaccine preparation became 0.2 ml/ml, thereby obtaining a mixture. The obtained mixture was stirred at 4° C. for 5 hours so as to adsorb the FFA on the aluminum phosphate gel. The resultant mixture was subjected to centrifugation at 2000 rpm for 20 minutes at 4° C. to collect the gel. The collected gel was suspended in 1/75 M phosphate buffer (pH 6.5). To the resultant suspension were added sucrose, L-arginine and Haemaccel (manufactured and sold by Hoechst Aktiengesellschaft, Germany) in this order in amounts such that the final concentrations of sucrose, L-arginine and Haemaccel became 3% (w/v), 1% (w/v) and 2% (w/v), respectively, followed by mixing to obtain an adsorbed FFA tetanus vaccine preparation. The obtained preparation was dispensed in glass vials having a volume of 1 ml so that each vial contained 0.6 ml of the preparation, and then, the vials were sealed. The obtained adsorbed FFA tetanus vaccine preparation was subjected to various tests in accordance with a provision entitled “adsorbed tetanus toxoid” in the Notification No. 217 of the Japanese Ministry of Health and Welfare: “Seibutsugakuteki Seizai Kijun (Minimum Requirements for Biological Products)”. As a result, the obtained preparation was verified as a qualified vaccine.

EXAMPLE 4

Production of an Adsorbed DPT Combined Vaccine Preparation Using an FFA Tetanus Vaccine

An adsorbed FFA vaccine was prepared in substantially the same manner as in Example 3, except that the final protein concentration of the adsorbed FFA tetanus vaccine was changed to 180 μg/ml. An adsorbed diphtheria toxoid and an adsorbed pertussis vaccine were individually prepared so that each of the toxoid concentration and the vaccine concentration became three times that of the working concentration. The adsorbed FFA tetanus vaccine, the adsorbed diphtheria toxoid and the adsorbed pertussis vaccine were mixed together to obtain an adsorbed DPT combined vaccine preparation. The obtained preparation was dispensed in glass vials each having a volume of 10 ml so that each vial contained 10 ml of the preparation, and then, the vials were sealed. The adsorbed DPT combined vaccine preparation was subjected to various tests in accordance with a provision entitled “adsorbed diphtheria-pertussis-tetanus combined vaccine” in the Notification No. 217 of the Japanese Ministry of Health and Welfare: “Seibutsugakuteki Seizai Kijun (Minimum Requirements for Biological Products)”. As a result, the obtained preparation was verified as a qualified combined vaccine.

EXAMPLE 5

Production of an Adsorbed DT Combined Vaccine Preparation Using an FFA Tetanus Vaccine

An adsorbed FFA tetanus vaccine was prepared in substantially the same manner as in Example 3, except that the final protein concentration of the adsorbed FFA tetanus vaccine was changed to 120 μg/ml. An adsorbed diphtheria toxoid was prepared so that the toxoid concentration became two times that of the working concentration. The adsorbed FFA tetanus vaccine and the diphtheria toxoid were mixed together to obtain an adsorbed DT combined vaccine preparation. The obtained preparation was dispensed in glass vials each having a volume of 1 ml so that each vial contained 0.6 ml of the preparation, and then, the vials were sealed. The obtained adsorbed DT combined vaccine preparation was subjected to various tests in accordance with a provision entitled “adsorbed diphtheria-tetanus combined vaccine” in the Notification No. 217 of the Japanese Ministry of Health and Welfare: “Seibutsugakuteki Seizai Kijun (Minimum Requirements for Biological Products)”. As a result, the obtained preparation was verified as a qualified combined vaccine.

EXAMPLE 6

Production of a Dried FFA Tetanus Vaccine Preparation

An FFA tetanus vaccine preparation was prepared in substantially the same manner as in Example 2. The obtained FFA tetanus vaccine preparation was dispensed in glass vials each having a volume of 1 ml so that each vial contained 0.6 ml of the preparation, followed by freeze-drying to obtain a dried FFA tetanus vaccine preparation. Then, the vials were sealed. One of the vials was unsealed and the dried FFA tetanus vaccine preparation was dissolved by sterilized distilled water so as to obtain 0.6 ml of vaccine solution, and the obtained vaccine solution was subjected to various tests in accordance with a provision entitled “tetanus toxoid” in the Notification No. 217 of the Japanese Ministry of Health and Welfare “Seibutsugakuteki Seizai Kijun (Minimum Requirements for Biological Products)”. As a result, the obtained preparation was verified as a qualified combined vaccine.

EXAMPLE 7

Production of a Sample FFA Tetanus Vaccine and Evaluation of the Immunopotency thereof

The production of the vaccine using the extracellular toxin and the evaluation of the immunopotency of the produced vaccine were conducted in substantially the same manner as in Example 1, except that the conditions employed were changed as follows.

The time for culturing the seed culture of C. tetani at 35° C. was changed to 6 days. The preparation of the FFA from the solution of the whole tetanus toxin molecule (nicked form) obtained by gel filtration using Ultrogel AcA 34 column was conducted as follows. A DTT-Tris buffer (see Reference Example 8) was added to the above-mentioned solution of the whole tetanus toxin molecule in an amount of 1 ml per 2 mg of the solution of the whole tetanus toxin molecule, followed by mixing. Then, the reaction was performed at 25° C. for 60 minutes to reduce the disulfide bridge present in the toxin molecule. Subsequently, the resultant reaction mixture was treated with urea by addition of a solid urea in an amount such that the final urea concentration became 4 M. The reaction mixture was applied to PD10 column (manufactured and sold by Pharmacia Biotech, Sweden) equilibrated with the Buffer A (0.2 mM Tris-HCl containing 2 M urea solution and 1 mM DTT; pH 7.0) and the elution was carried out using the above-mentioned Buffer A to replace the DTT-Tris buffer in the reaction mixture with the Buffer A. The resultant eluate containing dissociated toxin was subjected to column chromatography using Mono Q column (manufactured and sold by Pharmacia Biotech, Sweden) equilibrated with Buffer A, wherein the elution was carried out using FPLC (manufactured and sold by Pharmacia Biotech, Sweden) and Buffer B (formed by adding NaCl to Buffer A, wherein the NaCl concentration is increased by a linear gradient of from 0 to 0.5 M). With respect to the obtained eluate, analysis was made in the same manner as in Example 1. As a result, it was found that, among the fractions of the eluate exhibiting a peak at 280 nm, the fraction obtained earliest was the FFA.

The stabilization of the FFA was conducted by incubating the FFA solution in a mixture of 0.067 M phosphate buffer (Na-K, pH 7.8) containing 0.2% (v/v) of formalin, and 0.025 M lysine at 35° C. for 2 weeks. The obtained stabilized FFA was used to prepare the FFA tetanus vaccine.

The evaluation of the immunopotency of the sample FFA tetanus vaccine was conducted by 4 sets of an experiment using mice in substantially the same manner as in item (5) of Example 1. Results are shown in Table 3. As can be clearly seen from Table 3, the immunopotency of the sample FFA tetanus vaccine had substantially the same level as that of the control vaccine comprising the whole tetanus toxin toxoid (i. e., a conventional tetanus toxoid).

TABLE 1 Relative Fiducial limit Sample toxoid immunopotency (p = 0.95) Purified whole 1.0  toxin toxoid FFA 0.970 0.636-1.478 Purified whole toxin toxoid: Vaccine comprising the whole tetanus toxin toxoid FFA: Tetanus vaccine comprising the FFA

TABLE 2 Antigen used for sensitization and the degree of Amount of intradermal reaction antigen administered Conven- Purified by intradermal tional whole teta- injection (μg) toxoid nus toxoid FFA Conventional 3.2 +6 +6 +3 toxoid 1.0 +6 +5 +2 0.32 +5 +5 +1 Purified 3.2 +6 +6 +1 whole 1.0 +4 +5 +1 tetanus 0.32 +4 +4 *0 toxoid FFA 3.2 +5 +5 +1 1.0 +3 +3 *0 0.32 +1 +2 *0 Control 0 *0 *0 *0 Conventional toxoid: Commercially available tetanus toxoid Purified whole tetanus toxoid: Vaccine comprising a whole tetanus toxin toxoid FFA: Tetanus vaccine comprising the FFA

The degree of intradermal reaction is shown, using an indication selected from seven indications (*0) to (+6), in accordance with the following criteria, based on the size (E) of the erythema wherein E is a value of formula: ${\begin{matrix} {{major}\quad {diameter}\quad ({mm})} \\ {{of}\quad {the}\quad {erythema}} \end{matrix} \times \begin{matrix} {{minor}\quad {diameter}\quad ({mm})} \\ {{of}\quad {the}\quad {erythema}} \end{matrix}}:\begin{matrix} {{\,^{*}0}:} & {\left( {0 \leqq E < 1} \right),{{+ 1}:\left( {1 \leqq E < 20} \right)},{{+ 2}:\left( {20 \leqq E < 40} \right)},} \\ {{+ 3}:} & {\left( {40 \leqq E < 60} \right),{{+ 4}:\left( {60 \leqq E < 80} \right)},} \\ {{+ 5}:} & {\left( {80 \leqq E < 100} \right),{{{and} + 6}:{\left( {100 \leqq E} \right).}}} \end{matrix}$

TABLE 3 Relative Fiducial limits Sample toxoid immunopotency (p = 0.95) Purified whole 1.0 toxin toxid FFA  1.158 0.563-2.382 Purified whole toxin toxoid: Vaccine comprising the whole tetanus toxin toxoid FFA: Tetanus vaccine comprising the FFA

INDUSTRIAL APPLICABILITY

According to the present invention, an FFA (tetanus toxin functional fragment antigen) for a tetanus vaccine is provided, which is advantageous not only in that it is extremely excellent with respect to the diminution of adverse side effects, as compared to the conventional tetanus toxoids but also in that it has an immunopotency which is substantially the same as that of a conventional tetanus toxoid.

By the use of the FFA of the present invention as an active component for a tetanus vaccine, there can be provided a tetanus vaccine which is not only extremely excellent with respect to the diminution of adverse side effects, as compared to a conventional tetanus toxoid vaccine, but also has an immunopotency which is substantially the same as that of a conventional tetanus toxoid vaccine.

Further, the above-mentioned tetanus vaccine can also be provided in the form of a combined vaccine comprising the tetanus vaccine and at least one vaccine other than the tetanus vaccine, such as a pertussis vaccine and a diphtheria vaccine.

17 1 1315 PRT Clostridium tetani 1 Met Pro Ile Thr Ile Asn Asn Phe Arg Tyr Ser Asp Pro Val Asn Asn 1 5 10 15 Asp Thr Ile Ile Met Met Glu Pro Pro Tyr Cys Lys Gly Leu Asp Ile 20 25 30 Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Val Pro Glu 35 40 45 Arg Tyr Glu Phe Gly Thr Lys Pro Glu Asp Phe Asn Pro Pro Ser Ser 50 55 60 Leu Ile Glu Gly Ala Ser Glu Tyr Tyr Asp Pro Asn Tyr Leu Arg Thr 65 70 75 80 Asp Ser Asp Lys Asp Arg Phe Leu Gln Thr Met Val Lys Leu Phe Asn 85 90 95 Arg Ile Lys Asn Asn Val Ala Gly Glu Ala Leu Leu Asp Lys Ile Ile 100 105 110 Asn Ala Ile Pro Tyr Leu Gly Asn Ser Tyr Ser Leu Leu Asp Lys Phe 115 120 125 Asp Thr Asn Ser Asn Ser Val Ser Phe Asn Leu Leu Glu Gln Asp Pro 130 135 140 Ser Gly Ala Thr Thr Lys Ser Ala Met Leu Thr Asn Leu Ile Ile Phe 145 150 155 160 Gly Pro Gly Pro Val Leu Asn Lys Asn Glu Val Arg Gly Ile Val Leu 165 170 175 Arg Val Asp Asn Lys Asn Tyr Phe Pro Cys Arg Asp Gly Phe Gly Ser 180 185 190 Ile Met Gln Met Ala Phe Cys Pro Glu Tyr Val Pro Thr Phe Asp Asn 195 200 205 Val Ile Glu Asn Ile Thr Ser Leu Thr Ile Gly Lys Ser Lys Tyr Phe 210 215 220 Gln Asp Pro Ala Leu Leu Leu Met His Glu Leu Ile His Val Leu His 225 230 235 240 Gly Leu Tyr Gly Met Gln Val Ser Ser His Glu Ile Ile Pro Ser Lys 245 250 255 Gln Glu Ile Tyr Met Gln His Thr Tyr Pro Ile Ser Ala Glu Glu Leu 260 265 270 Phe Thr Phe Gly Gly Gln Asp Ala Asn Leu Ile Ser Ile Asp Ile Lys 275 280 285 Asn Asp Leu Tyr Glu Lys Thr Leu Asn Asp Tyr Lys Ala Ile Ala Asn 290 295 300 Lys Leu Ser Gln Val Thr Ser Cys Asn Asp Pro Asn Ile Asp Ile Asp 305 310 315 320 Ser Tyr Lys Gln Ile Tyr Gln Gln Lys Tyr Gln Phe Asp Lys Asp Ser 325 330 335 Asn Gly Gln Tyr Ile Val Asn Glu Asp Lys Phe Gln Ile Leu Tyr Asn 340 345 350 Ser Ile Met Tyr Gly Phe Thr Glu Ile Glu Leu Gly Lys Lys Phe Asn 355 360 365 Ile Lys Thr Arg Leu Ser Tyr Phe Ser Met Asn His Asp Pro Val Lys 370 375 380 Ile Pro Asn Leu Leu Asp Asp Thr Ile Tyr Asn Asp Thr Glu Gly Phe 385 390 395 400 Asn Ile Glu Ser Lys Asp Leu Lys Ser Glu Tyr Lys Gly Gln Asn Met 405 410 415 Arg Val Asn Thr Asn Ala Phe Arg Asn Val Asp Gly Ser Gly Leu Val 420 425 430 Ser Lys Leu Ile Gly Leu Cys Lys Lys Ile Ile Pro Pro Thr Asn Ile 435 440 445 Arg Glu Asn Leu Tyr Asn Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly 450 455 460 Glu Leu Cys Ile Lys Ile Lys Asn Glu Asp Leu Thr Phe Ile Ala Glu 465 470 475 480 Lys Asn Ser Phe Ser Glu Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr 485 490 495 Asn Thr Lys Asn Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile 500 505 510 Ile Val Asp Tyr Asn Leu Gln Ser Lys Ile Thr Leu Pro Asn Asp Arg 515 520 525 Thr Thr Pro Val Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser 530 535 540 Asn Ala Ala Ser Thr Ile Glu Ile His Asn Ile Asp Asp Asn Thr Ile 545 550 555 560 Tyr Gln Tyr Leu Tyr Ala Gln Lys Ser Pro Thr Thr Leu Gln Arg Ile 565 570 575 Thr Met Thr Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile 580 585 590 Tyr Ser Tyr Phe Pro Ser Val Ile Ser Lys Val Asn Gln Gly Ala Gln 595 600 605 Gly Ile Leu Phe Leu Gln Trp Val Arg Asp Ile Ile Asp Asp Phe Thr 610 615 620 Asn Glu Ser Ser Gln Lys Thr Thr Ile Asp Lys Ile Ser Asp Val Ser 625 630 635 640 Thr Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly 645 650 655 Tyr Glu Gly Asn Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu 660 665 670 Leu Leu Glu Tyr Ile Pro Glu Ile Thr Leu Pro Val Ile Ala Ala Leu 675 680 685 Ser Ile Ala Glu Ser Ser Thr Gln Lys Glu Lys Ile Ile Lys Thr Ile 690 695 700 Asp Asn Phe Leu Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val Tyr Lys 705 710 715 720 Leu Val Lys Ala Lys Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys 725 730 735 Arg Ser Tyr Gln Met Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile 740 745 750 Lys Lys Ile Ile Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Lys 755 760 765 Glu Gln Ile Ala Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu 770 775 780 Lys Ala Asn Lys Ala Met Ile Asn Ile Asn Ile Phe Met Arg Glu Ser 785 790 795 800 Ser Arg Ser Phe Leu Val Asn Gln Met Ile Asn Glu Ala Lys Lys Gln 805 810 815 Leu Leu Glu Phe Asp Thr Gln Ser Lys Asn Ile Leu Met Gln Tyr Ile 820 825 830 Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Glu 835 840 845 Ser Lys Ile Asn Lys Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser 850 855 860 Lys Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile 865 870 875 880 Leu Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile 885 890 895 Ser Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala 900 905 910 Gln Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn 915 920 925 Glu Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn 930 935 940 Asp Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys 945 950 955 960 Val Ser Ala Ser His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile 965 970 975 Ile Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser 980 985 990 Val Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala 995 1000 1005 Gly Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe Asn 1010 1015 1020 Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn Asp Arg 1025 1030 1035 1040 Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met Gly Ser Ala 1045 1050 1055 Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn Ile Thr Leu 1060 1065 1070 Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr Val Ser Ile Asp Lys 1075 1080 1085 Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys Glu Ile Glu Lys Leu 1090 1095 1100 Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu Arg Asp Phe Trp Gly Asn 1105 1110 1115 1120 Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr Leu Ile Pro Val Ala Ser Ser 1125 1130 1135 Ser Lys Asp Val Gln Leu Lys Asn Ile Thr Asp Tyr Met Tyr Leu Thr 1140 1145 1150 Asn Ala Pro Ser Tyr Thr Asn Gly Lys Leu Asn Ile Tyr Tyr Arg Arg 1155 1160 1165 Leu Tyr Asn Gly Leu Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn 1170 1175 1180 Glu Ile Asp Ser Phe Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val 1185 1190 1195 1200 Ser Tyr Asn Asn Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn 1205 1210 1215 Ala Phe Asn Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro 1220 1225 1230 Gly Ile Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu 1235 1240 1245 Lys Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala Ser 1250 1255 1260 Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp Pro Asn 1265 1270 1275 1280 Arg Asp Ile Leu Ile Ala Ser Asn Trp Tyr Phe Asn His Leu Lys Asp 1285 1290 1295 Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp Glu Gly Trp 1300 1305 1310 Thr Asn Asp 1315 2 29 PRT Clostridium tetani 2 Lys Ile Ile Pro Pro Thr Asn Ile Arg Glu Asn Leu Tyr Asn Arg Thr 1 5 10 15 Ala Ser Leu Thr Asp Leu Gly Gly Glu Leu Cys Ile Lys 20 25 3 28 PRT Clostridium tetani 3 Ile Ile Pro Pro Thr Asn Ile Arg Glu Asn Leu Tyr Asn Arg Thr Ala 1 5 10 15 Ser Leu Thr Asp Leu Gly Gly Glu Leu Cys Ile Lys 20 25 4 20 PRT Clostridium tetani 4 Glu Asn Leu Tyr Asn Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly Glu 1 5 10 15 Leu Cys Ile Lys 20 5 19 PRT Clostridium tetani 5 Asn Leu Tyr Asn Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly Glu Leu 1 5 10 15 Cys Ile Lys 6 16 PRT Clostridium tetani 6 Asn Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly Glu Leu Cys Ile Lys 1 5 10 15 7 14 PRT Clostridium tetani 7 Thr Ala Ser Leu Thr Asp Leu Gly Gly Glu Leu Cys Ile Lys 1 5 10 8 12 PRT Clostridium tetani 8 Ser Leu Thr Asp Leu Gly Gly Glu Leu Cys Ile Lys 1 5 10 9 7 PRT Clostridium tetani 9 Gly Gly Glu Leu Cys Ile Lys 1 5 10 875 PRT Clostridium tetani Amino Acids 441 to 1315 of SEQ ID NO 1 10 Lys Ile Ile Pro Pro Thr Asn Ile Arg Glu Asn Leu Tyr Asn Arg Thr 1 5 10 15 Ala Ser Leu Thr Asp Leu Gly Gly Glu Leu Cys Ile Lys Ile Lys Asn 20 25 30 Glu Asp Leu Thr Phe Ile Ala Glu Lys Asn Ser Phe Ser Glu Glu Pro 35 40 45 Phe Gln Asp Glu Ile Val Ser Tyr Asn Thr Lys Asn Lys Pro Leu Asn 50 55 60 Phe Asn Tyr Ser Leu Asp Lys Ile Ile Val Asp Tyr Asn Leu Gln Ser 65 70 75 80 Lys Ile Thr Leu Pro Asn Asp Arg Thr Thr Pro Val Thr Lys Gly Ile 85 90 95 Pro Tyr Ala Pro Glu Tyr Lys Ser Asn Ala Ala Ser Thr Ile Glu Ile 100 105 110 His Asn Ile Asp Asp Asn Thr Ile Tyr Gln Tyr Leu Tyr Ala Gln Lys 115 120 125 Ser Pro Thr Thr Leu Gln Arg Ile Thr Met Thr Asn Ser Val Asp Asp 130 135 140 Ala Leu Ile Asn Ser Thr Lys Ile Tyr Ser Tyr Phe Pro Ser Val Ile 145 150 155 160 Ser Lys Val Asn Gln Gly Ala Gln Gly Ile Leu Phe Leu Gln Trp Val 165 170 175 Arg Asp Ile Ile Asp Asp Phe Thr Asn Glu Ser Ser Gln Lys Thr Thr 180 185 190 Ile Asp Lys Ile Ser Asp Val Ser Thr Ile Val Pro Tyr Ile Gly Pro 195 200 205 Ala Leu Asn Ile Val Lys Gln Gly Tyr Glu Gly Asn Phe Ile Gly Ala 210 215 220 Leu Glu Thr Thr Gly Val Val Leu Leu Leu Glu Tyr Ile Pro Glu Ile 225 230 235 240 Thr Leu Pro Val Ile Ala Ala Leu Ser Ile Ala Glu Ser Ser Thr Gln 245 250 255 Lys Glu Lys Ile Ile Lys Thr Ile Asp Asn Phe Leu Glu Lys Arg Tyr 260 265 270 Glu Lys Trp Ile Glu Val Tyr Lys Leu Val Lys Ala Lys Trp Leu Gly 275 280 285 Thr Val Asn Thr Gln Phe Gln Lys Arg Ser Tyr Gln Met Tyr Arg Ser 290 295 300 Leu Glu Tyr Gln Val Asp Ala Ile Lys Lys Ile Ile Asp Tyr Glu Tyr 305 310 315 320 Lys Ile Tyr Ser Gly Pro Asp Lys Glu Gln Ile Ala Asp Glu Ile Asn 325 330 335 Asn Leu Lys Asn Lys Leu Glu Glu Lys Ala Asn Lys Ala Met Ile Asn 340 345 350 Ile Asn Ile Phe Met Arg Glu Ser Ser Arg Ser Phe Leu Val Asn Gln 355 360 365 Met Ile Asn Glu Ala Lys Lys Gln Leu Leu Glu Phe Asp Thr Gln Ser 370 375 380 Lys Asn Ile Leu Met Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly 385 390 395 400 Ile Thr Glu Leu Lys Lys Leu Glu Ser Lys Ile Asn Lys Val Phe Ser 405 410 415 Thr Pro Ile Pro Phe Ser Tyr Ser Lys Asn Leu Asp Cys Trp Val Asp 420 425 430 Asn Glu Glu Asp Ile Asp Val Ile Leu Lys Lys Ser Thr Ile Leu Asn 435 440 445 Leu Asp Ile Asn Asn Asp Ile Ile Ser Asp Ile Ser Gly Phe Asn Ser 450 455 460 Ser Val Ile Thr Tyr Pro Asp Ala Gln Leu Val Pro Gly Ile Asn Gly 465 470 475 480 Lys Ala Ile His Leu Val Asn Asn Glu Ser Ser Glu Val Ile Val His 485 490 495 Lys Ala Met Asp Ile Glu Tyr Asn Asp Met Phe Asn Asn Phe Thr Val 500 505 510 Ser Phe Trp Leu Arg Val Pro Lys Val Ser Ala Ser His Leu Glu Gln 515 520 525 Tyr Gly Thr Asn Glu Tyr Ser Ile Ile Ser Ser Met Lys Lys His Ser 530 535 540 Leu Ser Ile Gly Ser Gly Trp Ser Val Ser Leu Lys Gly Asn Asn Leu 545 550 555 560 Ile Trp Thr Leu Lys Asp Ser Ala Gly Glu Val Arg Gln Ile Thr Phe 565 570 575 Arg Asp Leu Pro Asp Lys Phe Asn Ala Tyr Leu Ala Asn Lys Trp Val 580 585 590 Phe Ile Thr Ile Thr Asn Asp Arg Leu Ser Ser Ala Asn Leu Tyr Ile 595 600 605 Asn Gly Val Leu Met Gly Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile 610 615 620 Arg Glu Asp Asn Asn Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn 625 630 635 640 Asn Gln Tyr Val Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu 645 650 655 Asn Pro Lys Glu Ile Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr 660 665 670 Phe Leu Arg Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr 675 680 685 Tyr Leu Ile Pro Val Ala Ser Ser Ser Lys Asp Val Gln Leu Lys Asn 690 695 700 Ile Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Ser Tyr Thr Asn Gly 705 710 715 720 Lys Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu Lys Phe Ile 725 730 735 Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Ser Phe Val Lys Ser 740 745 750 Gly Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn Asn Asn Glu His Ile 755 760 765 Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe Asn Asn Leu Asp Arg Ile 770 775 780 Leu Arg Val Gly Tyr Asn Ala Pro Gly Ile Pro Leu Tyr Lys Lys Met 785 790 795 800 Glu Ala Val Lys Leu Arg Asp Leu Lys Thr Tyr Ser Val Gln Leu Lys 805 810 815 Leu Tyr Asp Asp Lys Asn Ala Ser Leu Gly Leu Val Gly Thr His Asn 820 825 830 Gly Gln Ile Gly Asn Asp Pro Asn Arg Asp Ile Leu Ile Ala Ser Asn 835 840 845 Trp Tyr Phe Asn His Leu Lys Asp Lys Ile Leu Gly Cys Asp Trp Tyr 850 855 860 Phe Val Pro Thr Asp Glu Gly Trp Thr Asn Asp 865 870 875 11 874 PRT Clostridium tetani Amino Acids 442 to 1315 of SEQ ID NO 1 11 Ile Ile Pro Pro Thr Asn Ile Arg Glu Asn Leu Tyr Asn Arg Thr Ala 1 5 10 15 Ser Leu Thr Asp Leu Gly Gly Glu Leu Cys Ile Lys Ile Lys Asn Glu 20 25 30 Asp Leu Thr Phe Ile Ala Glu Lys Asn Ser Phe Ser Glu Glu Pro Phe 35 40 45 Gln Asp Glu Ile Val Ser Tyr Asn Thr Lys Asn Lys Pro Leu Asn Phe 50 55 60 Asn Tyr Ser Leu Asp Lys Ile Ile Val Asp Tyr Asn Leu Gln Ser Lys 65 70 75 80 Ile Thr Leu Pro Asn Asp Arg Thr Thr Pro Val Thr Lys Gly Ile Pro 85 90 95 Tyr Ala Pro Glu Tyr Lys Ser Asn Ala Ala Ser Thr Ile Glu Ile His 100 105 110 Asn Ile Asp Asp Asn Thr Ile Tyr Gln Tyr Leu Tyr Ala Gln Lys Ser 115 120 125 Pro Thr Thr Leu Gln Arg Ile Thr Met Thr Asn Ser Val Asp Asp Ala 130 135 140 Leu Ile Asn Ser Thr Lys Ile Tyr Ser Tyr Phe Pro Ser Val Ile Ser 145 150 155 160 Lys Val Asn Gln Gly Ala Gln Gly Ile Leu Phe Leu Gln Trp Val Arg 165 170 175 Asp Ile Ile Asp Asp Phe Thr Asn Glu Ser Ser Gln Lys Thr Thr Ile 180 185 190 Asp Lys Ile Ser Asp Val Ser Thr Ile Val Pro Tyr Ile Gly Pro Ala 195 200 205 Leu Asn Ile Val Lys Gln Gly Tyr Glu Gly Asn Phe Ile Gly Ala Leu 210 215 220 Glu Thr Thr Gly Val Val Leu Leu Leu Glu Tyr Ile Pro Glu Ile Thr 225 230 235 240 Leu Pro Val Ile Ala Ala Leu Ser Ile Ala Glu Ser Ser Thr Gln Lys 245 250 255 Glu Lys Ile Ile Lys Thr Ile Asp Asn Phe Leu Glu Lys Arg Tyr Glu 260 265 270 Lys Trp Ile Glu Val Tyr Lys Leu Val Lys Ala Lys Trp Leu Gly Thr 275 280 285 Val Asn Thr Gln Phe Gln Lys Arg Ser Tyr Gln Met Tyr Arg Ser Leu 290 295 300 Glu Tyr Gln Val Asp Ala Ile Lys Lys Ile Ile Asp Tyr Glu Tyr Lys 305 310 315 320 Ile Tyr Ser Gly Pro Asp Lys Glu Gln Ile Ala Asp Glu Ile Asn Asn 325 330 335 Leu Lys Asn Lys Leu Glu Glu Lys Ala Asn Lys Ala Met Ile Asn Ile 340 345 350 Asn Ile Phe Met Arg Glu Ser Ser Arg Ser Phe Leu Val Asn Gln Met 355 360 365 Ile Asn Glu Ala Lys Lys Gln Leu Leu Glu Phe Asp Thr Gln Ser Lys 370 375 380 Asn Ile Leu Met Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile 385 390 395 400 Thr Glu Leu Lys Lys Leu Glu Ser Lys Ile Asn Lys Val Phe Ser Thr 405 410 415 Pro Ile Pro Phe Ser Tyr Ser Lys Asn Leu Asp Cys Trp Val Asp Asn 420 425 430 Glu Glu Asp Ile Asp Val Ile Leu Lys Lys Ser Thr Ile Leu Asn Leu 435 440 445 Asp Ile Asn Asn Asp Ile Ile Ser Asp Ile Ser Gly Phe Asn Ser Ser 450 455 460 Val Ile Thr Tyr Pro Asp Ala Gln Leu Val Pro Gly Ile Asn Gly Lys 465 470 475 480 Ala Ile His Leu Val Asn Asn Glu Ser Ser Glu Val Ile Val His Lys 485 490 495 Ala Met Asp Ile Glu Tyr Asn Asp Met Phe Asn Asn Phe Thr Val Ser 500 505 510 Phe Trp Leu Arg Val Pro Lys Val Ser Ala Ser His Leu Glu Gln Tyr 515 520 525 Gly Thr Asn Glu Tyr Ser Ile Ile Ser Ser Met Lys Lys His Ser Leu 530 535 540 Ser Ile Gly Ser Gly Trp Ser Val Ser Leu Lys Gly Asn Asn Leu Ile 545 550 555 560 Trp Thr Leu Lys Asp Ser Ala Gly Glu Val Arg Gln Ile Thr Phe Arg 565 570 575 Asp Leu Pro Asp Lys Phe Asn Ala Tyr Leu Ala Asn Lys Trp Val Phe 580 585 590 Ile Thr Ile Thr Asn Asp Arg Leu Ser Ser Ala Asn Leu Tyr Ile Asn 595 600 605 Gly Val Leu Met Gly Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg 610 615 620 Glu Asp Asn Asn Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn 625 630 635 640 Gln Tyr Val Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn 645 650 655 Pro Lys Glu Ile Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe 660 665 670 Leu Arg Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr 675 680 685 Leu Ile Pro Val Ala Ser Ser Ser Lys Asp Val Gln Leu Lys Asn Ile 690 695 700 Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Ser Tyr Thr Asn Gly Lys 705 710 715 720 Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu Lys Phe Ile Ile 725 730 735 Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Ser Phe Val Lys Ser Gly 740 745 750 Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn Asn Asn Glu His Ile Val 755 760 765 Gly Tyr Pro Lys Asp Gly Asn Ala Phe Asn Asn Leu Asp Arg Ile Leu 770 775 780 Arg Val Gly Tyr Asn Ala Pro Gly Ile Pro Leu Tyr Lys Lys Met Glu 785 790 795 800 Ala Val Lys Leu Arg Asp Leu Lys Thr Tyr Ser Val Gln Leu Lys Leu 805 810 815 Tyr Asp Asp Lys Asn Ala Ser Leu Gly Leu Val Gly Thr His Asn Gly 820 825 830 Gln Ile Gly Asn Asp Pro Asn Arg Asp Ile Leu Ile Ala Ser Asn Trp 835 840 845 Tyr Phe Asn His Leu Lys Asp Lys Ile Leu Gly Cys Asp Trp Tyr Phe 850 855 860 Val Pro Thr Asp Glu Gly Trp Thr Asn Asp 865 870 12 866 PRT Clostridium tetani Amino Acids 450 to 1315 of SEQ ID NO 1 12 Glu Asn Leu Tyr Asn Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly Glu 1 5 10 15 Leu Cys Ile Lys Ile Lys Asn Glu Asp Leu Thr Phe Ile Ala Glu Lys 20 25 30 Asn Ser Phe Ser Glu Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr Asn 35 40 45 Thr Lys Asn Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile Ile 50 55 60 Val Asp Tyr Asn Leu Gln Ser Lys Ile Thr Leu Pro Asn Asp Arg Thr 65 70 75 80 Thr Pro Val Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser Asn 85 90 95 Ala Ala Ser Thr Ile Glu Ile His Asn Ile Asp Asp Asn Thr Ile Tyr 100 105 110 Gln Tyr Leu Tyr Ala Gln Lys Ser Pro Thr Thr Leu Gln Arg Ile Thr 115 120 125 Met Thr Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile Tyr 130 135 140 Ser Tyr Phe Pro Ser Val Ile Ser Lys Val Asn Gln Gly Ala Gln Gly 145 150 155 160 Ile Leu Phe Leu Gln Trp Val Arg Asp Ile Ile Asp Asp Phe Thr Asn 165 170 175 Glu Ser Ser Gln Lys Thr Thr Ile Asp Lys Ile Ser Asp Val Ser Thr 180 185 190 Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly Tyr 195 200 205 Glu Gly Asn Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu Leu 210 215 220 Leu Glu Tyr Ile Pro Glu Ile Thr Leu Pro Val Ile Ala Ala Leu Ser 225 230 235 240 Ile Ala Glu Ser Ser Thr Gln Lys Glu Lys Ile Ile Lys Thr Ile Asp 245 250 255 Asn Phe Leu Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val Tyr Lys Leu 260 265 270 Val Lys Ala Lys Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys Arg 275 280 285 Ser Tyr Gln Met Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile Lys 290 295 300 Lys Ile Ile Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Lys Glu 305 310 315 320 Gln Ile Ala Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu Lys 325 330 335 Ala Asn Lys Ala Met Ile Asn Ile Asn Ile Phe Met Arg Glu Ser Ser 340 345 350 Arg Ser Phe Leu Val Asn Gln Met Ile Asn Glu Ala Lys Lys Gln Leu 355 360 365 Leu Glu Phe Asp Thr Gln Ser Lys Asn Ile Leu Met Gln Tyr Ile Lys 370 375 380 Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Glu Ser 385 390 395 400 Lys Ile Asn Lys Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser Lys 405 410 415 Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile Leu 420 425 430 Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile Ser 435 440 445 Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala Gln 450 455 460 Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn Glu 465 470 475 480 Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn Asp 485 490 495 Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val 500 505 510 Ser Ala Ser His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile Ile 515 520 525 Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser Val 530 535 540 Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala Gly 545 550 555 560 Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe Asn Ala 565 570 575 Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn Asp Arg Leu 580 585 590 Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met Gly Ser Ala Glu 595 600 605 Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn Ile Thr Leu Lys 610 615 620 Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr Val Ser Ile Asp Lys Phe 625 630 635 640 Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys Glu Ile Glu Lys Leu Tyr 645 650 655 Thr Ser Tyr Leu Ser Ile Thr Phe Leu Arg Asp Phe Trp Gly Asn Pro 660 665 670 Leu Arg Tyr Asp Thr Glu Tyr Tyr Leu Ile Pro Val Ala Ser Ser Ser 675 680 685 Lys Asp Val Gln Leu Lys Asn Ile Thr Asp Tyr Met Tyr Leu Thr Asn 690 695 700 Ala Pro Ser Tyr Thr Asn Gly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu 705 710 715 720 Tyr Asn Gly Leu Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu 725 730 735 Ile Asp Ser Phe Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val Ser 740 745 750 Tyr Asn Asn Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala 755 760 765 Phe Asn Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro Gly 770 775 780 Ile Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu Lys 785 790 795 800 Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala Ser Leu 805 810 815 Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp Pro Asn Arg 820 825 830 Asp Ile Leu Ile Ala Ser Asn Trp Tyr Phe Asn His Leu Lys Asp Lys 835 840 845 Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp Glu Gly Trp Thr 850 855 860 Asn Asp 865 13 865 PRT Clostridium tetani Amino Acids 451 to 1315 of SEQ ID NO 1 13 Asn Leu Tyr Asn Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly Glu Leu 1 5 10 15 Cys Ile Lys Ile Lys Asn Glu Asp Leu Thr Phe Ile Ala Glu Lys Asn 20 25 30 Ser Phe Ser Glu Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr Asn Thr 35 40 45 Lys Asn Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile Ile Val 50 55 60 Asp Tyr Asn Leu Gln Ser Lys Ile Thr Leu Pro Asn Asp Arg Thr Thr 65 70 75 80 Pro Val Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser Asn Ala 85 90 95 Ala Ser Thr Ile Glu Ile His Asn Ile Asp Asp Asn Thr Ile Tyr Gln 100 105 110 Tyr Leu Tyr Ala Gln Lys Ser Pro Thr Thr Leu Gln Arg Ile Thr Met 115 120 125 Thr Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile Tyr Ser 130 135 140 Tyr Phe Pro Ser Val Ile Ser Lys Val Asn Gln Gly Ala Gln Gly Ile 145 150 155 160 Leu Phe Leu Gln Trp Val Arg Asp Ile Ile Asp Asp Phe Thr Asn Glu 165 170 175 Ser Ser Gln Lys Thr Thr Ile Asp Lys Ile Ser Asp Val Ser Thr Ile 180 185 190 Val Pro Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly Tyr Glu 195 200 205 Gly Asn Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu Leu Leu 210 215 220 Glu Tyr Ile Pro Glu Ile Thr Leu Pro Val Ile Ala Ala Leu Ser Ile 225 230 235 240 Ala Glu Ser Ser Thr Gln Lys Glu Lys Ile Ile Lys Thr Ile Asp Asn 245 250 255 Phe Leu Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val Tyr Lys Leu Val 260 265 270 Lys Ala Lys Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys Arg Ser 275 280 285 Tyr Gln Met Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile Lys Lys 290 295 300 Ile Ile Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Lys Glu Gln 305 310 315 320 Ile Ala Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu Lys Ala 325 330 335 Asn Lys Ala Met Ile Asn Ile Asn Ile Phe Met Arg Glu Ser Ser Arg 340 345 350 Ser Phe Leu Val Asn Gln Met Ile Asn Glu Ala Lys Lys Gln Leu Leu 355 360 365 Glu Phe Asp Thr Gln Ser Lys Asn Ile Leu Met Gln Tyr Ile Lys Ala 370 375 380 Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Glu Ser Lys 385 390 395 400 Ile Asn Lys Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser Lys Asn 405 410 415 Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile Leu Lys 420 425 430 Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile Ser Asp 435 440 445 Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala Gln Leu 450 455 460 Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn Glu Ser 465 470 475 480 Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn Asp Met 485 490 495 Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser 500 505 510 Ala Ser His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile Ile Ser 515 520 525 Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser Val Ser 530 535 540 Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala Gly Glu 545 550 555 560 Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe Asn Ala Tyr 565 570 575 Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn Asp Arg Leu Ser 580 585 590 Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met Gly Ser Ala Glu Ile 595 600 605 Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn Ile Thr Leu Lys Leu 610 615 620 Asp Arg Cys Asn Asn Asn Asn Gln Tyr Val Ser Ile Asp Lys Phe Arg 625 630 635 640 Ile Phe Cys Lys Ala Leu Asn Pro Lys Glu Ile Glu Lys Leu Tyr Thr 645 650 655 Ser Tyr Leu Ser Ile Thr Phe Leu Arg Asp Phe Trp Gly Asn Pro Leu 660 665 670 Arg Tyr Asp Thr Glu Tyr Tyr Leu Ile Pro Val Ala Ser Ser Ser Lys 675 680 685 Asp Val Gln Leu Lys Asn Ile Thr Asp Tyr Met Tyr Leu Thr Asn Ala 690 695 700 Pro Ser Tyr Thr Asn Gly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr 705 710 715 720 Asn Gly Leu Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile 725 730 735 Asp Ser Phe Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val Ser Tyr 740 745 750 Asn Asn Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe 755 760 765 Asn Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro Gly Ile 770 775 780 Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu Lys Thr 785 790 795 800 Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala Ser Leu Gly 805 810 815 Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp Pro Asn Arg Asp 820 825 830 Ile Leu Ile Ala Ser Asn Trp Tyr Phe Asn His Leu Lys Asp Lys Ile 835 840 845 Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp Glu Gly Trp Thr Asn 850 855 860 Asp 865 14 862 PRT Clostridium tetani Amino Acids 454 to 1315 of SEQ ID NO 1 14 Asn Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly Glu Leu Cys Ile Lys 1 5 10 15 Ile Lys Asn Glu Asp Leu Thr Phe Ile Ala Glu Lys Asn Ser Phe Ser 20 25 30 Glu Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr Asn Thr Lys Asn Lys 35 40 45 Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile Ile Val Asp Tyr Asn 50 55 60 Leu Gln Ser Lys Ile Thr Leu Pro Asn Asp Arg Thr Thr Pro Val Thr 65 70 75 80 Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser Asn Ala Ala Ser Thr 85 90 95 Ile Glu Ile His Asn Ile Asp Asp Asn Thr Ile Tyr Gln Tyr Leu Tyr 100 105 110 Ala Gln Lys Ser Pro Thr Thr Leu Gln Arg Ile Thr Met Thr Asn Ser 115 120 125 Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile Tyr Ser Tyr Phe Pro 130 135 140 Ser Val Ile Ser Lys Val Asn Gln Gly Ala Gln Gly Ile Leu Phe Leu 145 150 155 160 Gln Trp Val Arg Asp Ile Ile Asp Asp Phe Thr Asn Glu Ser Ser Gln 165 170 175 Lys Thr Thr Ile Asp Lys Ile Ser Asp Val Ser Thr Ile Val Pro Tyr 180 185 190 Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly Tyr Glu Gly Asn Phe 195 200 205 Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu Leu Leu Glu Tyr Ile 210 215 220 Pro Glu Ile Thr Leu Pro Val Ile Ala Ala Leu Ser Ile Ala Glu Ser 225 230 235 240 Ser Thr Gln Lys Glu Lys Ile Ile Lys Thr Ile Asp Asn Phe Leu Glu 245 250 255 Lys Arg Tyr Glu Lys Trp Ile Glu Val Tyr Lys Leu Val Lys Ala Lys 260 265 270 Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys Arg Ser Tyr Gln Met 275 280 285 Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile Lys Lys Ile Ile Asp 290 295 300 Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Lys Glu Gln Ile Ala Asp 305 310 315 320 Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu Lys Ala Asn Lys Ala 325 330 335 Met Ile Asn Ile Asn Ile Phe Met Arg Glu Ser Ser Arg Ser Phe Leu 340 345 350 Val Asn Gln Met Ile Asn Glu Ala Lys Lys Gln Leu Leu Glu Phe Asp 355 360 365 Thr Gln Ser Lys Asn Ile Leu Met Gln Tyr Ile Lys Ala Asn Ser Lys 370 375 380 Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Glu Ser Lys Ile Asn Lys 385 390 395 400 Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser Lys Asn Leu Asp Cys 405 410 415 Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile Leu Lys Lys Ser Thr 420 425 430 Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile Ser Asp Ile Ser Gly 435 440 445 Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala Gln Leu Val Pro Gly 450 455 460 Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn Glu Ser Ser Glu Val 465 470 475 480 Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn Asp Met Phe Asn Asn 485 490 495 Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser Ala Ser His 500 505 510 Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile Ile Ser Ser Met Lys 515 520 525 Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser Val Ser Leu Lys Gly 530 535 540 Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala Gly Glu Val Arg Gln 545 550 555 560 Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe Asn Ala Tyr Leu Ala Asn 565 570 575 Lys Trp Val Phe Ile Thr Ile Thr Asn Asp Arg Leu Ser Ser Ala Asn 580 585 590 Leu Tyr Ile Asn Gly Val Leu Met Gly Ser Ala Glu Ile Thr Gly Leu 595 600 605 Gly Ala Ile Arg Glu Asp Asn Asn Ile Thr Leu Lys Leu Asp Arg Cys 610 615 620 Asn Asn Asn Asn Gln Tyr Val Ser Ile Asp Lys Phe Arg Ile Phe Cys 625 630 635 640 Lys Ala Leu Asn Pro Lys Glu Ile Glu Lys Leu Tyr Thr Ser Tyr Leu 645 650 655 Ser Ile Thr Phe Leu Arg Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp 660 665 670 Thr Glu Tyr Tyr Leu Ile Pro Val Ala Ser Ser Ser Lys Asp Val Gln 675 680 685 Leu Lys Asn Ile Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Ser Tyr 690 695 700 Thr Asn Gly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu 705 710 715 720 Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Ser Phe 725 730 735 Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn Asn Asn 740 745 750 Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe Asn Asn Leu 755 760 765 Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro Gly Ile Pro Leu Tyr 770 775 780 Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu Lys Thr Tyr Ser Val 785 790 795 800 Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala Ser Leu Gly Leu Val Gly 805 810 815 Thr His Asn Gly Gln Ile Gly Asn Asp Pro Asn Arg Asp Ile Leu Ile 820 825 830 Ala Ser Asn Trp Tyr Phe Asn His Leu Lys Asp Lys Ile Leu Gly Cys 835 840 845 Asp Trp Tyr Phe Val Pro Thr Asp Glu Gly Trp Thr Asn Asp 850 855 860 15 860 PRT Clostridium tetani Amino Acids 456 to 1315 of SEQ ID NO 1 15 Thr Ala Ser Leu Thr Asp Leu Gly Gly Glu Leu Cys Ile Lys Ile Lys 1 5 10 15 Asn Glu Asp Leu Thr Phe Ile Ala Glu Lys Asn Ser Phe Ser Glu Glu 20 25 30 Pro Phe Gln Asp Glu Ile Val Ser Tyr Asn Thr Lys Asn Lys Pro Leu 35 40 45 Asn Phe Asn Tyr Ser Leu Asp Lys Ile Ile Val Asp Tyr Asn Leu Gln 50 55 60 Ser Lys Ile Thr Leu Pro Asn Asp Arg Thr Thr Pro Val Thr Lys Gly 65 70 75 80 Ile Pro Tyr Ala Pro Glu Tyr Lys Ser Asn Ala Ala Ser Thr Ile Glu 85 90 95 Ile His Asn Ile Asp Asp Asn Thr Ile Tyr Gln Tyr Leu Tyr Ala Gln 100 105 110 Lys Ser Pro Thr Thr Leu Gln Arg Ile Thr Met Thr Asn Ser Val Asp 115 120 125 Asp Ala Leu Ile Asn Ser Thr Lys Ile Tyr Ser Tyr Phe Pro Ser Val 130 135 140 Ile Ser Lys Val Asn Gln Gly Ala Gln Gly Ile Leu Phe Leu Gln Trp 145 150 155 160 Val Arg Asp Ile Ile Asp Asp Phe Thr Asn Glu Ser Ser Gln Lys Thr 165 170 175 Thr Ile Asp Lys Ile Ser Asp Val Ser Thr Ile Val Pro Tyr Ile Gly 180 185 190 Pro Ala Leu Asn Ile Val Lys Gln Gly Tyr Glu Gly Asn Phe Ile Gly 195 200 205 Ala Leu Glu Thr Thr Gly Val Val Leu Leu Leu Glu Tyr Ile Pro Glu 210 215 220 Ile Thr Leu Pro Val Ile Ala Ala Leu Ser Ile Ala Glu Ser Ser Thr 225 230 235 240 Gln Lys Glu Lys Ile Ile Lys Thr Ile Asp Asn Phe Leu Glu Lys Arg 245 250 255 Tyr Glu Lys Trp Ile Glu Val Tyr Lys Leu Val Lys Ala Lys Trp Leu 260 265 270 Gly Thr Val Asn Thr Gln Phe Gln Lys Arg Ser Tyr Gln Met Tyr Arg 275 280 285 Ser Leu Glu Tyr Gln Val Asp Ala Ile Lys Lys Ile Ile Asp Tyr Glu 290 295 300 Tyr Lys Ile Tyr Ser Gly Pro Asp Lys Glu Gln Ile Ala Asp Glu Ile 305 310 315 320 Asn Asn Leu Lys Asn Lys Leu Glu Glu Lys Ala Asn Lys Ala Met Ile 325 330 335 Asn Ile Asn Ile Phe Met Arg Glu Ser Ser Arg Ser Phe Leu Val Asn 340 345 350 Gln Met Ile Asn Glu Ala Lys Lys Gln Leu Leu Glu Phe Asp Thr Gln 355 360 365 Ser Lys Asn Ile Leu Met Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile 370 375 380 Gly Ile Thr Glu Leu Lys Lys Leu Glu Ser Lys Ile Asn Lys Val Phe 385 390 395 400 Ser Thr Pro Ile Pro Phe Ser Tyr Ser Lys Asn Leu Asp Cys Trp Val 405 410 415 Asp Asn Glu Glu Asp Ile Asp Val Ile Leu Lys Lys Ser Thr Ile Leu 420 425 430 Asn Leu Asp Ile Asn Asn Asp Ile Ile Ser Asp Ile Ser Gly Phe Asn 435 440 445 Ser Ser Val Ile Thr Tyr Pro Asp Ala Gln Leu Val Pro Gly Ile Asn 450 455 460 Gly Lys Ala Ile His Leu Val Asn Asn Glu Ser Ser Glu Val Ile Val 465 470 475 480 His Lys Ala Met Asp Ile Glu Tyr Asn Asp Met Phe Asn Asn Phe Thr 485 490 495 Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser Ala Ser His Leu Glu 500 505 510 Gln Tyr Gly Thr Asn Glu Tyr Ser Ile Ile Ser Ser Met Lys Lys His 515 520 525 Ser Leu Ser Ile Gly Ser Gly Trp Ser Val Ser Leu Lys Gly Asn Asn 530 535 540 Leu Ile Trp Thr Leu Lys Asp Ser Ala Gly Glu Val Arg Gln Ile Thr 545 550 555 560 Phe Arg Asp Leu Pro Asp Lys Phe Asn Ala Tyr Leu Ala Asn Lys Trp 565 570 575 Val Phe Ile Thr Ile Thr Asn Asp Arg Leu Ser Ser Ala Asn Leu Tyr 580 585 590 Ile Asn Gly Val Leu Met Gly Ser Ala Glu Ile Thr Gly Leu Gly Ala 595 600 605 Ile Arg Glu Asp Asn Asn Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn 610 615 620 Asn Asn Gln Tyr Val Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala 625 630 635 640 Leu Asn Pro Lys Glu Ile Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile 645 650 655 Thr Phe Leu Arg Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu 660 665 670 Tyr Tyr Leu Ile Pro Val Ala Ser Ser Ser Lys Asp Val Gln Leu Lys 675 680 685 Asn Ile Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Ser Tyr Thr Asn 690 695 700 Gly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu Lys Phe 705 710 715 720 Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Ser Phe Val Lys 725 730 735 Ser Gly Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn Asn Asn Glu His 740 745 750 Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe Asn Asn Leu Asp Arg 755 760 765 Ile Leu Arg Val Gly Tyr Asn Ala Pro Gly Ile Pro Leu Tyr Lys Lys 770 775 780 Met Glu Ala Val Lys Leu Arg Asp Leu Lys Thr Tyr Ser Val Gln Leu 785 790 795 800 Lys Leu Tyr Asp Asp Lys Asn Ala Ser Leu Gly Leu Val Gly Thr His 805 810 815 Asn Gly Gln Ile Gly Asn Asp Pro Asn Arg Asp Ile Leu Ile Ala Ser 820 825 830 Asn Trp Tyr Phe Asn His Leu Lys Asp Lys Ile Leu Gly Cys Asp Trp 835 840 845 Tyr Phe Val Pro Thr Asp Glu Gly Trp Thr Asn Asp 850 855 860 16 858 PRT Clostridium tetani Amino Acids 458 to 1315 of SEQ ID NO 1 16 Ser Leu Thr Asp Leu Gly Gly Glu Leu Cys Ile Lys Ile Lys Asn Glu 1 5 10 15 Asp Leu Thr Phe Ile Ala Glu Lys Asn Ser Phe Ser Glu Glu Pro Phe 20 25 30 Gln Asp Glu Ile Val Ser Tyr Asn Thr Lys Asn Lys Pro Leu Asn Phe 35 40 45 Asn Tyr Ser Leu Asp Lys Ile Ile Val Asp Tyr Asn Leu Gln Ser Lys 50 55 60 Ile Thr Leu Pro Asn Asp Arg Thr Thr Pro Val Thr Lys Gly Ile Pro 65 70 75 80 Tyr Ala Pro Glu Tyr Lys Ser Asn Ala Ala Ser Thr Ile Glu Ile His 85 90 95 Asn Ile Asp Asp Asn Thr Ile Tyr Gln Tyr Leu Tyr Ala Gln Lys Ser 100 105 110 Pro Thr Thr Leu Gln Arg Ile Thr Met Thr Asn Ser Val Asp Asp Ala 115 120 125 Leu Ile Asn Ser Thr Lys Ile Tyr Ser Tyr Phe Pro Ser Val Ile Ser 130 135 140 Lys Val Asn Gln Gly Ala Gln Gly Ile Leu Phe Leu Gln Trp Val Arg 145 150 155 160 Asp Ile Ile Asp Asp Phe Thr Asn Glu Ser Ser Gln Lys Thr Thr Ile 165 170 175 Asp Lys Ile Ser Asp Val Ser Thr Ile Val Pro Tyr Ile Gly Pro Ala 180 185 190 Leu Asn Ile Val Lys Gln Gly Tyr Glu Gly Asn Phe Ile Gly Ala Leu 195 200 205 Glu Thr Thr Gly Val Val Leu Leu Leu Glu Tyr Ile Pro Glu Ile Thr 210 215 220 Leu Pro Val Ile Ala Ala Leu Ser Ile Ala Glu Ser Ser Thr Gln Lys 225 230 235 240 Glu Lys Ile Ile Lys Thr Ile Asp Asn Phe Leu Glu Lys Arg Tyr Glu 245 250 255 Lys Trp Ile Glu Val Tyr Lys Leu Val Lys Ala Lys Trp Leu Gly Thr 260 265 270 Val Asn Thr Gln Phe Gln Lys Arg Ser Tyr Gln Met Tyr Arg Ser Leu 275 280 285 Glu Tyr Gln Val Asp Ala Ile Lys Lys Ile Ile Asp Tyr Glu Tyr Lys 290 295 300 Ile Tyr Ser Gly Pro Asp Lys Glu Gln Ile Ala Asp Glu Ile Asn Asn 305 310 315 320 Leu Lys Asn Lys Leu Glu Glu Lys Ala Asn Lys Ala Met Ile Asn Ile 325 330 335 Asn Ile Phe Met Arg Glu Ser Ser Arg Ser Phe Leu Val Asn Gln Met 340 345 350 Ile Asn Glu Ala Lys Lys Gln Leu Leu Glu Phe Asp Thr Gln Ser Lys 355 360 365 Asn Ile Leu Met Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile 370 375 380 Thr Glu Leu Lys Lys Leu Glu Ser Lys Ile Asn Lys Val Phe Ser Thr 385 390 395 400 Pro Ile Pro Phe Ser Tyr Ser Lys Asn Leu Asp Cys Trp Val Asp Asn 405 410 415 Glu Glu Asp Ile Asp Val Ile Leu Lys Lys Ser Thr Ile Leu Asn Leu 420 425 430 Asp Ile Asn Asn Asp Ile Ile Ser Asp Ile Ser Gly Phe Asn Ser Ser 435 440 445 Val Ile Thr Tyr Pro Asp Ala Gln Leu Val Pro Gly Ile Asn Gly Lys 450 455 460 Ala Ile His Leu Val Asn Asn Glu Ser Ser Glu Val Ile Val His Lys 465 470 475 480 Ala Met Asp Ile Glu Tyr Asn Asp Met Phe Asn Asn Phe Thr Val Ser 485 490 495 Phe Trp Leu Arg Val Pro Lys Val Ser Ala Ser His Leu Glu Gln Tyr 500 505 510 Gly Thr Asn Glu Tyr Ser Ile Ile Ser Ser Met Lys Lys His Ser Leu 515 520 525 Ser Ile Gly Ser Gly Trp Ser Val Ser Leu Lys Gly Asn Asn Leu Ile 530 535 540 Trp Thr Leu Lys Asp Ser Ala Gly Glu Val Arg Gln Ile Thr Phe Arg 545 550 555 560 Asp Leu Pro Asp Lys Phe Asn Ala Tyr Leu Ala Asn Lys Trp Val Phe 565 570 575 Ile Thr Ile Thr Asn Asp Arg Leu Ser Ser Ala Asn Leu Tyr Ile Asn 580 585 590 Gly Val Leu Met Gly Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg 595 600 605 Glu Asp Asn Asn Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn 610 615 620 Gln Tyr Val Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn 625 630 635 640 Pro Lys Glu Ile Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe 645 650 655 Leu Arg Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr 660 665 670 Leu Ile Pro Val Ala Ser Ser Ser Lys Asp Val Gln Leu Lys Asn Ile 675 680 685 Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Ser Tyr Thr Asn Gly Lys 690 695 700 Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu Lys Phe Ile Ile 705 710 715 720 Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Ser Phe Val Lys Ser Gly 725 730 735 Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn Asn Asn Glu His Ile Val 740 745 750 Gly Tyr Pro Lys Asp Gly Asn Ala Phe Asn Asn Leu Asp Arg Ile Leu 755 760 765 Arg Val Gly Tyr Asn Ala Pro Gly Ile Pro Leu Tyr Lys Lys Met Glu 770 775 780 Ala Val Lys Leu Arg Asp Leu Lys Thr Tyr Ser Val Gln Leu Lys Leu 785 790 795 800 Tyr Asp Asp Lys Asn Ala Ser Leu Gly Leu Val Gly Thr His Asn Gly 805 810 815 Gln Ile Gly Asn Asp Pro Asn Arg Asp Ile Leu Ile Ala Ser Asn Trp 820 825 830 Tyr Phe Asn His Leu Lys Asp Lys Ile Leu Gly Cys Asp Trp Tyr Phe 835 840 845 Val Pro Thr Asp Glu Gly Trp Thr Asn Asp 850 855 17 853 PRT Clostridium tetani Amino Acids 463 to 1315 of SEQ ID NO 1 17 Gly Gly Glu Leu Cys Ile Lys Ile Lys Asn Glu Asp Leu Thr Phe Ile 1 5 10 15 Ala Glu Lys Asn Ser Phe Ser Glu Glu Pro Phe Gln Asp Glu Ile Val 20 25 30 Ser Tyr Asn Thr Lys Asn Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp 35 40 45 Lys Ile Ile Val Asp Tyr Asn Leu Gln Ser Lys Ile Thr Leu Pro Asn 50 55 60 Asp Arg Thr Thr Pro Val Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr 65 70 75 80 Lys Ser Asn Ala Ala Ser Thr Ile Glu Ile His Asn Ile Asp Asp Asn 85 90 95 Thr Ile Tyr Gln Tyr Leu Tyr Ala Gln Lys Ser Pro Thr Thr Leu Gln 100 105 110 Arg Ile Thr Met Thr Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr 115 120 125 Lys Ile Tyr Ser Tyr Phe Pro Ser Val Ile Ser Lys Val Asn Gln Gly 130 135 140 Ala Gln Gly Ile Leu Phe Leu Gln Trp Val Arg Asp Ile Ile Asp Asp 145 150 155 160 Phe Thr Asn Glu Ser Ser Gln Lys Thr Thr Ile Asp Lys Ile Ser Asp 165 170 175 Val Ser Thr Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys 180 185 190 Gln Gly Tyr Glu Gly Asn Phe Ile Gly Ala Leu Glu Thr Thr Gly Val 195 200 205 Val Leu Leu Leu Glu Tyr Ile Pro Glu Ile Thr Leu Pro Val Ile Ala 210 215 220 Ala Leu Ser Ile Ala Glu Ser Ser Thr Gln Lys Glu Lys Ile Ile Lys 225 230 235 240 Thr Ile Asp Asn Phe Leu Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val 245 250 255 Tyr Lys Leu Val Lys Ala Lys Trp Leu Gly Thr Val Asn Thr Gln Phe 260 265 270 Gln Lys Arg Ser Tyr Gln Met Tyr Arg Ser Leu Glu Tyr Gln Val Asp 275 280 285 Ala Ile Lys Lys Ile Ile Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro 290 295 300 Asp Lys Glu Gln Ile Ala Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu 305 310 315 320 Glu Glu Lys Ala Asn Lys Ala Met Ile Asn Ile Asn Ile Phe Met Arg 325 330 335 Glu Ser Ser Arg Ser Phe Leu Val Asn Gln Met Ile Asn Glu Ala Lys 340 345 350 Lys Gln Leu Leu Glu Phe Asp Thr Gln Ser Lys Asn Ile Leu Met Gln 355 360 365 Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys 370 375 380 Leu Glu Ser Lys Ile Asn Lys Val Phe Ser Thr Pro Ile Pro Phe Ser 385 390 395 400 Tyr Ser Lys Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp 405 410 415 Val Ile Leu Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp 420 425 430 Ile Ile Ser Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro 435 440 445 Asp Ala Gln Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val 450 455 460 Asn Asn Glu Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu 465 470 475 480 Tyr Asn Asp Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val 485 490 495 Pro Lys Val Ser Ala Ser His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr 500 505 510 Ser Ile Ile Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly 515 520 525 Trp Ser Val Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp 530 535 540 Ser Ala Gly Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys 545 550 555 560 Phe Asn Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn 565 570 575 Asp Arg Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met Gly 580 585 590 Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn Ile 595 600 605 Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr Val Ser Ile 610 615 620 Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys Glu Ile Glu 625 630 635 640 Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu Arg Asp Phe Trp 645 650 655 Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr Leu Ile Pro Val Ala 660 665 670 Ser Ser Ser Lys Asp Val Gln Leu Lys Asn Ile Thr Asp Tyr Met Tyr 675 680 685 Leu Thr Asn Ala Pro Ser Tyr Thr Asn Gly Lys Leu Asn Ile Tyr Tyr 690 695 700 Arg Arg Leu Tyr Asn Gly Leu Lys Phe Ile Ile Lys Arg Tyr Thr Pro 705 710 715 720 Asn Asn Glu Ile Asp Ser Phe Val Lys Ser Gly Asp Phe Ile Lys Leu 725 730 735 Tyr Val Ser Tyr Asn Asn Asn Glu His Ile Val Gly Tyr Pro Lys Asp 740 745 750 Gly Asn Ala Phe Asn Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn 755 760 765 Ala Pro Gly Ile Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg 770 775 780 Asp Leu Lys Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn 785 790 795 800 Ala Ser Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp 805 810 815 Pro Asn Arg Asp Ile Leu Ile Ala Ser Asn Trp Tyr Phe Asn His Leu 820 825 830 Lys Asp Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp Glu 835 840 845 Gly Trp Thr Asn Asp 850 

What is claimed is:
 1. A tetanus toxin functional fragment antigen which is stabilized with a fixative, wherein the tetanus toxin functional fragment antigen prior to stabilization with said fixative comprises at least one independent fragment which is obtained by a process comprising the steps of splitting at least one peptide bond selected from peptide bonds individually connecting mutually adjacent amino acid residues in a partial amino acid sequence between two cysteine residues participating in forming a disulfide bridge present in the N-terminal of the entire amino acid sequence of a whole tetanus toxin molecule, splitting said disulfide bridge, and splitting non-covalent bonds between groups of the tetanus toxin molecule; said tetanus toxin functional fragment antigen prior to stabilization with said fixative having: (a) a molecular weight of from 90000 to 110000 as measured by an SDS-polyacrylamide gel electrophoresis method; and (b) an isoelectric point of 7.25±0.5 as measured by an isoelectric focusing method, wherein each of said at least one independent fragment has an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17.
 2. A combined vaccine comprising the tetanus toxin functional fragment antigen of claim 1 and at least one additional antigen selected from the group consisting of diphtheria toxoid and an antigen for pertussis vaccine, each in an effective immunogenic amount.
 3. A method for producing a tetanus vaccine, which comprises stabilizing a tetanus toxin functional fragment antigen with a fixative, said tetanus toxin functional fragment antigen comprising at least one independent fragment which is obtained by a process comprising the steps of collecting and purifying an extracellular tetanus toxin from a culture filtrate of Clostridium tetani to obtain an extracellular tetanus toxin molecule, splitting a disulfide bridge of said extracellular tetanus toxin molecule, and splitting non-covalent bonds between groups on the extracellular tetanus toxin molecule; said tetanus toxin functional fragment antigen having: (a) a molecular weight of from 90000 to 110000 as measured by an SDS-polyacrylamide gel electrophoresis method; and (b) an isoelectric point of 7.25±0.5 as measured by an isoelectric focusing method, wherein each of said at least one independent fragment has an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17.
 4. A tetanus vaccine comprising, as an active component, the tetanus toxin functional fragment antigen of claim 1 in an effective immunogenic amount. 